Functions and Mechanisms of Receptor Tyrosine Kinase Torso Signaling: Lessons From Drosophila Embryonic Terminal Development

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1 DEVELOPMENTAL DYNAMICS 232: , 2005 REVIEWS A PEER REVIEWED FORUM Functions and Mechanisms of Receptor Tyrosine Kinase Torso Signaling: Lessons From Drosophila Embryonic Terminal Development Willis X. Li* The Torso receptor tyrosine kinase (RTK) is required for cell fate specification in the terminal regions (head and tail) of the early Drosophila embryo. Torso contains a split tyrosine kinase domain and belongs to the type III subgroup of the RTK superfamily that also includes the platelet-derived growth factor receptors, stem cell or steel factor receptor c-kit proto-oncoprotein, colony-stimulating factor-1 receptor, and vascular endothelial growth factor receptor. The Torso pathway has been a model system for studying RTK signal transduction. Genetic and biochemical studies of Torso signaling have provided valuable insights into the biological functions and mechanisms of RTK signaling during early Drosophila embryogenesis. Developmental Dynamics 232: , Wiley-Liss, Inc. Key words: Torso; Drosophila; review; type III receptor tyrosine kinase; embryonic development; terminal system; PDGF receptor; c-kit Received 30 August 2004; Revised 26 October 2004; Accepted 27 October 2004 INTRODUCTION Torso is a maternally contributed receptor tyrosine kinase (RTK), i.e., it is made by the nurse cells during oogenesis and deposited into the oocyte, which gives rise to the mature egg (Casanova and Struhl, 1989; Sprenger et al., 1989; reviewed by Duffy and Perrimon, 1994; Furriols and Casanova, 2003). Torso is the earliest known RTK to be activated during Drosophila embryogenesis. It is required for cell fate specification and pattern formation in Drosophila embryonic cells located in the anterior and posterior terminal regions of the embryo. Along with the anterior, posterior, and dorsoventral systems, the terminal signaling pathway is one of the four maternally contributed systems essential for setting up the initial coordinates of the egg, allowing progressive elaboration of the embryonic body plan. Torso is only transiently present in the early embryo and is not detected after gastrulation. In the early syncytial embryo, Torso is uniformly distributed on the membrane but is activated only at the two terminal regions by ligands whose production is spatially restricted to the termini (Casanova and Struhl, 1989; Sprenger et al., 1989; Stevens et al., 1990). Terminal cell fates, thus, are specified by localized activation of a uniformly distributed RTK (reviewed by St. Johnston and Nusslein- Volhard, 1992; Duffy and Perrimon, 1994; Furriols and Casanova, 2003). Aside from its role in pattern formation, the Torso pathway has been a paradigm for genetically dissecting RTK signaling mechanisms. Early genetic studies of Torso signal transduction mainly focused on isolating components mediating Torso signaling and determining their sequence of action. Genetic screens, epistatic analyses, and mosaic analyses produced valuable information in this regard. Genetic studies of Torso signaling have demonstrated that Torso transduces signals mainly by means of the Ras extracellular signal regulated ki- Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, New York Grant sponsor; National Institutes of Health, National Institute Of General Medical Sciences; Grant number: R01GM *Correspondence to: Willis X. Li, Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY willis_li@urmc.rochester.edu DOI /dvdy Published online 9 February 2005 in Wiley InterScience ( Wiley-Liss, Inc.

2 DROSOPHILA TORSO SIGNALING 657 nase (ERK) signaling cassette (reviewed by Lu et al., 1993b; Duffy and Perrimon, 1994; Perrimon et al., 1995), but it also activates STAT, which plays a role in mediating a subset of Torso signals (Li et al., 2002, 2003). Biochemical studies have revealed that the signaling strategies used by Torso are highly conserved among all RTKs (Cleghon et al., 1996, 1998). Several RTKs have been identified in Drosophila that play essential roles in distinct developmental processes. Besides Torso, two other well-studied Drosophila RTKs are the epidermal growth factor receptor (EGFR) and Sevenless. The Sevenless pathway, which signals by means of the Ras-ERK signaling cassette, is required for specifying the R7 photoreceptor cell fate during eye development (reviewed by Wassarman et al., 1995). The Drosophila EGFR homolog (also known as DER) acts through the same signaling cassette. It is used repeatedly by a variety of tissues during different stages of Drosophila development to regulate cell fate determination, cell proliferation, and cell migration (reviewed by Shilo, 2003). These RTKs share common signaling mediators and mainly use the canonical Ras- ERK pathway to transduce signals. The Torso pathway specifies cell fate by inducing target gene expression. Two of the best-known Torso target genes are tailless (tll) and huckebein (hkb), which encode nuclear hormone receptor and zinc-finger transcription factors, respectively (Pignoni et al., 1990; Weigel et al., 1990). In the embryonic posterior, Torso signaling induces the expression of tll and hkb, specifying the posterior terminal cell fates. In the anterior region, however, the combined activities of Torso and the anterior morphogen Bicoid determine the proper formation of the head (reviewed by Schmidt-Ott, 2001; Ephrussi and St. Johnston, 2004). Thus, the correct specification of terminal cell fates depends on developmentally controlled activation of the Torso signaling pathway, the participation of additional factors/pathways, and the developmental history of the cell nuclei. IDENTIFICATION OF TORSO After fertilization, the early Drosophila embryo undergoes 13 rapid synchronous nuclear divisions without cytokinesis, resulting in a syncytium, with a single cell body encompassing all of the nuclei (Bate and Martinez Arias, 1993; Campos-Ortega and Hartenstein, 1997). Each synchronous nuclear division cycle takes only 10 min on average. At the end of the 13th cycle, cellularization takes place, resulting in a cellular blastoderm. By this stage, all cells have acquired positional coordinates according to their spatial localization in the embryo. It had been postulated that the first steps of pattern formation and cell fate specification of a multicellular organism are determined by morphogen gradients that are set up by asymmetrical localization of maternal gene products in the early syncytium (Wolpert, 1996). Nusslein-Volhard and Wieschaus sought to identify systematically, by genetic screens, the maternal-effect genes required for patterning the Drosophila embryo (Schupbach and Wieschaus, 1986; Nusslein-Volhard et al., 1987). These genes encode products that are synthesized in the ovary of the mother and are directly deposited into the oocyte. One of the genes identified in these screens was torso. Genetic screens designed to isolate mutations affecting embryonic patterning identified three groups of maternal genes whose functions are essential for specifying cell fates along the anterior/posterior axis and another group specifying fates along the dorsal/ventral axis of the embryo (reviewed by St. Johnston and Nusslein- Volhard, 1992; Nusslein-Volhard, 2004) (Ephrussi and St. Johnston, 2004). Genes of the Bicoid group control pattern formation of the anterior region, including part of the head and the thorax. The Nanos group specifies cell fates in the abdominal region. The nonsegmented regions of the tail and part of the head are controlled by the Torso, or terminal, group of genes. The fourth group of maternal genes, the dorsal class, is essential for dorsoventral patterning. Together these four groups of maternal gene products act in the early embryo to define four sets of positional coordinates and, thus, begin to establish the body plan of the animal (reviewed by St. Johnston and Nusslein-Volhard, 1992; Nusslein-Volhard, 2004; Ephrussi and St. Johnston, 2004). As expected for a maternal gene, torso homozygous mutant flies are perfectly viable, but the females lay eggs that exhibit gross patterning defects they are missing the head and tail structures, hence the name torso. Mutations in other members of the terminal group genes show similar embryonic patterning defects: the embryos exhibit normal patterning of central structures but are missing tissues derived from the acron and telson (Duffy and Perrimon, 1994). In Drosophila, the acron refers to the anterior structures including the labrum and head skeleton. The telson includes the last (eighth) abdominal segment, posterior spiracles, Filzkörper, and Malphigian tubules (Fig. 1). TORSO IS A TYPE III RTK WITH A SPLIT TYROSINE KINASE DOMAIN Molecular investigations led to the discovery that Torso is an RTK of 923 amino acids that consists of an N-terminal extracellular region, a single transmembrane domain, and a C-terminal cytoplasmic tyrosine kinase domain (Casanova and Struhl, 1989; Sprenger et al., 1989). The extracellular domain, responsible for ligand binding, shows no homology to any known RTKs. Although full-length Torso is most homologous to the RET (REarranged during Transfection) proto-oncoprotein (reviewed by Jhiang, 2000), because the sequence homology includes the kinase domain and C-terminal tail (49% identical and 67% homologous, respectively), its split tyrosine kinase domain makes it structurally homologous to the type III RTKs (Fig. 2). Moreover, its kinase domain is significantly similar to those of the platelet-derived growth factor (PDGF) receptors and c-kit. A subgroup of RTKs (type III), including the PDGF receptors, c-kit, CSF-1R (also know as c-fms), FMSlike tyrosine kinase-3 (FLT-3), and vascular endothelial growth factor receptor (VEGF-R), share a common structural feature: the intracellular tyrosine kinase domain is split by an

3 658 LI Fig. 1. insert region comprising a stretch of approximately 100 hydrophobic amino acid residues (van der Geer et al., 1994; Hanks and Hunter, 1995). Torso shares this structural feature and, thus, belongs to this subgroup. In vertebrates, the type III RTKs are likely derived from a single ancestral RTK with a split tyrosine kinase domain for the following reasons. First, in mammals and pufferfish, genes for PDGF R and PDGF R are tandemly linked with c-kit and CSF1R, respectively, and share a similar genomic organization, suggesting that these genes are duplicates of an ancestral RTK gene (Roberts et al., 1988; Williams et al., 2002). Second, extensive sequence homology is evident along the whole molecule among type III RTKs. PDGF R is most similar to PDGF R, C-Kit, and CSF-1R, with sequence homology of 59%, 51%, 48%, Fig. 2. Fig. 3. Activation of Torso. Nasrat and Polehole are present uniformly on the outer plasma membrane of the embryo. Torso-like is associated to the inner surface of the vitelline membrane in the anterior and posterior pole. Trunk is processed and activated only at the poles by collective actions of Torso-like, Nasrat/Polehole, and an unidentified factor(s). Processed C-terminal fragment of Trunk activates Torso. Torso in turn impedes the further diffusion of active Trunk molecules. Fig. 1. Expression patterns of the Torso target gene tailless and cuticle phenotypes of two classes of torso mutants. Loss-of-function (lof) mutations in torso eliminate posterior tailless (tll) expression and posterior structures; gain-offunction (gof) mutations result in expansion of tll expression and ectopic and enlargement of posterior structures at the expense of central elements. Note the loss of Filzkörper (arrow in wild-type [WT] embryo) and A8 in the torso lof mutant embryo and deletion of central denticle belts and enlargement and ectopic Filzkörper in torso GOF embryo. Embryos are shown anterior to the left, posterior right. Dark blue stain in the left panels indicates tll mrna, as detected by in situ hybridization. Darkfield photographs of embryonic cuticular preparations are shown in the central panels, and schematic representations of these cuticular patterns are shown on the right. Red color indicates tissues derived from the acron (left) and telson (right). These tissues include the head skeletons (open arrows), the eighth abdominal denticle belt (A8), and the Filzkörper (closed arrows). A1 to A8, abdominal denticle belts; T1 to T3, thoracic segments. Fig. 2. Structure of receptor tyrosine kinases. Schematic outline of Torso and other receptor tyrosine kinases (RTKs). The type III RTK subfamily also includes c-kit, CSF-1R (also known as c-fms), and FMS-like tyrosine kinase-3 (FLT-3, not shown). These RTKs contain a kinase insert region with phosphotyrosine residues that serve as docking sites for downstream signaling molecules. This feature is not shared by other subfamily of RTKs, such as fibroblast growth factor receptor (FGF-R) and RET (REarranged during Transfection). The extracellular domain of Torso is not similar to the type III RTKs. PDGF-R, platelet-derived growth factor receptor; VEGF-R, vascular endothelial growth factor receptor; Ig, immunoglobulin.

4 DROSOPHILA TORSO SIGNALING 659 and 45%, respectively. It is also significantly homologous to FLT-3 and VEGF-R, although less similar in sequence. Outside of the type III RTK subgroup, the PDGF receptors are significantly homologous to the fibroblast growth factor (FGFR) receptors and RET, with sequence homology mainly restricted to the kinase domain. Third, certain signaling mechanisms are conserved within the type III subgroup of RTKs. For instance, many type III RTKs are capable of activating Ras-MAPK, STAT, PLC, and phosphatidylinositol 3-kinase (PI3-kinase) signaling (van der Geer et al., 1994). Although the role of Drosophila PI3K has not been evaluated with regard to Torso signaling, Torso is able to activate Ras and STAT92E (Li et al., 2003). Torso and the PDGF receptors are not only similar at the structural level, but they also share certain functional or signaling specificities. It has been shown recently that a PDGF R/ Torso fusion protein can fulfill certain functional requirements of PDGF R in knockin mice, whereas a similar fusion with mouse FGFR1 fails to rescue any aspect of the PDGF R null phenotype (Hamilton et al., 2003). Although the combined tyrosine kinase domain of Torso is more homologous to that of vertebrate bfgfr1 (52% identical and 72% similar) than to that of PDGF R (50% identical and 67% similar), results from the above in vivo experiments suggest that the structural similarity, i.e., the split kinase domain, is more important for the shared specificity between Torso and PDGF R. This finding suggests that the specificity of RTK signaling lies more in the particular downstream signaling molecules or adaptor proteins with which the receptor can associate than in the sequence of its tyrosine kinase domain. From comparisons with kinases of known structure, it is believed that the insert forms a loop that protrudes from the globular kinase domain surface (reviewed by van der Geer et al., 1994). Indeed, the kinase insert regions of the PDGF receptors and Torso contain many phosphotyrosine residues and play important roles in recruiting adaptor proteins (see below; reviewed by Heldin and Westermark, 1999). GENETIC STUDIES OF TORSO SIGNALING Identification of Torso Pathway Components by Genetic Screens The functions of a gene can best be inferred from its loss-of-function phenotypes. Genetic screens by Nusslein- Volhard, Wieschaus, and others identified several loci, referred to as the terminal or torso group genes, because when mutated they share similar embryonic phenotypes (reviewed by Duffy and Perrimon, 1994). The torso group genes initially included torso (tor), torso-like (tsl), trunk (trk), female sterile (1) Nasrat [fs(1)n], female sterile (1) pole hole [fs(1)ph], tll, and hkb (Degelmann et al., 1986; Perrimon et al., 1986; Nusslein-Volhard et al., 1987; Klingler et al., 1988; Casanova and Struhl, 1989; Sprenger et al., 1989; Stevens et al., 1990; Weigel et al., 1990). Mutations in the first five loci are female sterile and in the latter two are zygotic lethal. Mutations in these genes result in similar embryonic patterning defects; the embryos are missing the tissues derived from the acron and telson but the embryo has perfect central elements. The genetic screens conducted by Nusslein- Volhard and Wieschaus were saturated, because multiple alleles were recovered for most genes isolated. However, such genetic screens could not easily identify essential genes (those mutations would cause lethality) whose maternal products affect embryonic patterning or genes required at multiple developmental stages or with multiple functions. To study the maternal functions of zygotically essential genes, Perrimon s group used a dominant female sterile mutation, ovo D1, to produce germline mosaic females and isolated X-linked zygotic lethals with specific maternaleffect phenotypes (Perrimon et al., 1989; see below). This study identified lethal (1) pole hole [l(1)ph] and corkscrew (csw) as novel members of the torso class judging by the similarity of their loss-of-function phenotypes to those of torso mutant embryos. Later molecular studies revealed that l(1)ph and csw encode the Drosophila homologs of Raf and the SH2 domaincontaining tyrosine phosphatase (SHP2), respectively (Ambrosio et al., 1989b; Perkins et al., 1992). Epistatic Analyses Gain-of-function mutations are useful for determining the order of the components in a genetic pathway. Gainof-function alleles of torso (torso GOF ) cause patterning defects that appear to be the opposite of its loss-of-function phenotypes (Klingler et al., 1988; Schupbach and Wieschaus, 1989; Szabad et al., 1989). These embryos exhibit overgrown and ectopic terminal structures at the expense of central elements (Fig. 1). The expansion of terminal cell fates is associated with ectopic and higher levels of expression of the target gene tll (Fig. 1). Genetic epistatic analyses studies of phenotypes resulting from double mutants of torso GOF and a loss-of-function allele of the terminal class demonstrated that four of the terminal class genes act upstream of torso, whereas all the others function downstream. Specifically, mutations in tsl, trk, fs(1)ph, and fs(1)n do not affect torso GOF phenotypes and, thus, were postulated to be involved in the production of Torso ligands, whereas mutations in genes such as csw, Draf, and Dsor1, suppress torso GOF phenotypes, which is consistent with their being essential cytoplasmic transducers of Torso signals (Ambrosio et al., 1989b; Stevens et al., 1990; Perkins et al., 1992; Casanova and Struhl, 1993; Tsuda et al., 1993). Suppression of torso GOF phenotypes by mutations in another gene routinely has been taken to implicate the gene in Torso signaling. Use of Germ-Line Clone Embryos to Study Signaling Mechanisms Drosophila offers an advantageous genetic system for studying mechanisms of intracellular signal transduction. The Torso pathway operates during the early stages of embryogenesis to specify terminal cell fates. In the early embryo, all signaling components or other molecules are maternally contributed, i.e., they are made by the nurse cells during oogenesis and deposited into the oocyte, which gives rise to the mature egg. Importantly, for reasons not completely under-

5 660 LI stood, many essential signaling molecules, such as Ras1 and Draf, appear dispensable for oogenesis, such that eggs completely lacking these molecules are produced and can proceed to develop. This finding is in contrast to the somatic tissues where these molecules are required for cell viability and/or division. This particular aspect of Drosophila development allows one to study the effects of systematic removal of one or more gene products on embryonic development. The Flp-mediated dominant female sterility (Flp- DFS) technique developed in the Perrimon laboratory allows the generation of large quantities of embryos derived from germ cells homozygous for one or more particular mutations (Perrimon et al., 1989; Chou and Perrimon, 1992; Perrimon et al., 1996). These germ-line clone (GLC) embryos are equivalent to knockout animals for particular maternal gene products. The technique provides a powerful tool for genetic screens as well as for dissection of signaling pathways in early embryos (reviewed by Perrimon et al., 1995). An example is provided in the following section. Mechanisms of Raf Activation: Insights From GLC Embryos Despite extensive research efforts, the mechanisms underlying Raf activation still remain unclear. The Torso signaling system provides unique advantages by virtue of the simplicity and accessibility of Drosophila genetics: There are single copies of genes encoding components of the Ras-ERK signaling cassette, and the gene products can be totally removed, singly or in combination, from the early embryo by using the Flp-DFS technique, a manipulation that presently cannot be performed in any other organism. Many such analyses have been successfully performed to answer questions pertaining to Ras/Raf signaling (reviewed by Perrimon et al., 1995). Studies using Drosophila GLC embryos have provided mechanistic insights into the role of Ras in Raf activation. Unlike torso or Draf GLC embryos, in which no posterior tll expression can be detected, GLC embryos null for Ras1 still retain residual levels of tll expression in the posterior domain. Because Drosophila Ras2 is not redundant with Ras1, the data suggest that Raf can be activated by a Ras-independent mechanism (Hou et al., 1995). However, when the activities of several variant Draf proteins were examined in the complete absence of Ras1, it was found that all forms of Draf that had been believed previously to be constitutively active require Ras1 for activity (Li et al., 1998). These variant Draf proteins included N-terminally truncated Draf as well as the membrane targeted or both. Therefore, it appears that the role of Ras in Raf activation is not limited to membrane translocation of Raf through Ras-Raf association, a widely accepted model. Ras is also essential for the activation of an additional factor(s) that in turn activates Raf. Results from genetic analyses of Draf, thus, stand in contrast to studies on cultured cells, which have led to the proposal that the sole function of Ras in Raf activation is to bring it to the membrane (Leevers et al., 1994; Stokoe et al., 1994). Examination of a Ras null condition that can be achieved only in Drosophila GLC embryos has led to the conclusion that Ras has a novel second function, which is to activate a Raf activator that in turn activates Raf (Li et al., 1998; see Fig. 4). COMPONENTS OF THE TORSO PATHWAY Genetic studies have led to the conclusion that Torso and other known Drosophila RTKs share common signaling mediators and that they mainly use the canonical Ras-ERK pathway to transduce signals. Evidence that many of the same downstream components are required for transducing Torso signals was derived from examining their loss-of-function mutant phenotypes and epistatic relationships with torso GOF mutations. For example, removal of Draf from early embryos, by means of germline mosaics, results in phenotypes identical to those of torso null mutations (Ambrosio et al., 1989b; Hou et al., 1995). Similar phenotypes were also observed for Dsor1 (Tsuda et al., 1993). These results suggest that Torso signals may be mediated entirely by the Ras1/Draf/Dsor1/Rolled signaling cassette. Table 1 lists components of the Torso signaling pathway isolated and/or confirmed by the similarity of their loss-of-function phenotypes to those of torso mutations or by the ability of the mutations to suppress torso GOF phenotypes. Ligands: Activation of Torso Torso is uniformly distributed along the membrane of the early embryo but is activated in a spatially graded manner by diffusible ligands that are produced only at the terminal regions; their diffusion to other regions is impeded by interaction with Torso (reviewed by Furriols and Casanova, 2003; LeMosy, 2003). Trunk (Trk), a secreted protein synthesized by the oocyte during oogenesis (Schupbach and Wieschaus, 1986), has been proposed to be the Torso ligand (Casanova et al., 1995). Trk contains a cystine knot motif in its C-terminal region, a feature shared by certain growth factors and extracellular ligands. In particular, features of the Trk sequence resemble those of Spätzle (Spz), a secreted protein and proteolytically activated ligand for the dorsal ventral patterning receptor Toll (Morisato and Anderson, 1994; Casanova et al., 1995). Such similarity suggests that Trk might also be cleaved by proteolysis to generate an active Torso ligand. Indeed, a C-terminal fragment of Trk but not the whole molecule is sufficient to activate Torso in the absence of the other three genes normally required for Torso activation, namely, torso-like, fs(1)nasrat, and fs(1)ph (Savant-Bhonsale and Montell, 1993; Casali and Casanova, 2001). This finding has led to the hypothesis that Nasrat, Polehole, and Torsolike are involved in the proteolytic processing and activation of Trk. Protein products encoded by fs(1)nasrat and fs(1)ph accumulate at the oocyte surface during late stages of oogenesis and are essential for the cross-linking of vitelline membrane proteins, ensuring correct formation of the egg-shell as well as its integrity (Jimenez et al., 2000). In addition, Nasrat and Polehole are also essential for stabilizing Torsolike, which has been shown recently to be tethered to the inner surface of the vitelline membrane (Stevens et al., 2003). In the absence of Nasrat and Polehole, Torsolike is not efficiently localized to the

6 DROSOPHILA TORSO SIGNALING 661 poles of the early embryo (Jimenez et al., 2000; Stevens et al., 2003). Moreover, misexpression of Torsolike at different levels and in different genetic backgrounds suggests that Torsolike depends on Nasrat or Polehole not only for localization but also for activity; in addition, yet another unidentified factor at the poles is required for Torsolike function (Stevens et al., 2003; Fig. 3). Given all this complexity, evidence that Trk is actually cleaved proteolytically is still lacking, and the precise mechanism of how Nasrat, Polehole, and Torsolike cooperate to bring about Trk activation in conjunction with an unknown factor is still not clear. Cytoplasmic Mediators of Torso Signaling After ligand binding, RTKs dimerize and undergo autophosphorylation on several tyrosine residues. This process serves two purposes. First, phosphorylation of certain tyrosine residues within the kinase domain is required for the full activation of its enzymatic activity. Second, and more importantly, autophosphorylation of tyrosine residues outside the kinase domain creates docking sites for signal transducers or adaptors that contain Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains (reviewed by van der Geer et al., 1994; Pawson and Scott, 1997). Adaptors. Adaptor proteins play essential roles in mediating RTK signaling (reviewed by Pawson and Scott, 1997). The first adaptor identified in Drosophila was Downstream of receptor kinase (Drk), an SH2 SH3 adaptor protein and homolog of mammalian GRB2 and Caenorhabditis elegans Sem-5 (Clark et al., 1992; Lowenstein et al., 1992; Olivier et al., 1993; Simon et al., 1993). It was identified in a genetic screen for dosage-sensitive mediators of Sevenless signaling (Bonfini et al., 1992; Simon et al., 1993) and was later shown to be required for Torso signaling as well (Hou et al., 1995). Like GRB2, Drk binds to Son of Sevenless (Sos), a guanine nucleotide exchange factor for Ras, linking RTK activation to Ras signaling. Although Drk directly binds to Sevenless, its binding to other Drosophila RTKs is yet to be demonstrated (Raabe et al., 1996). In the case of Torso, Corkscrew (Csw) appears to mediate the association of Drk with the RTK (see below). Another adaptor identified in Drosophila in similar dosage-sensitive genetic screens is Daughter of Sevenless (DOS), a pleckstrin homology domaincontaining protein and a substrate of Csw (Herbst et al., 1996; Raabe et al., 1996). Finally, a Drosophila SHC adaptor protein (DSHC), first identified in by sequence homology, has been shown to mediate signaling of a subset of Drosophila RTKs: dshc mutations affect signaling by Torso and EGFR but not Sevenless (Lai et al., 1995; Luschnig et al., 2000). These adaptors may all be necessary or may function redundantly in mediating Torso signaling. Core signal transducers. The Ras/Raf/MEK/ERK (Ras1/Draf/ Dsor1/Rolled) signaling cassette appears to be the obligatory core signal transducer of all known Drosophila RTKs, including Torso, EGFR, Sevenless, and the FGFR homologs Heartless and Breathless (reviewed by Basler and Hafen, 1990; Perrimon, 1994). The Ras-ERK signaling cassette appears to be highly conserved evolutionarily throughout metazoans and plays a major role in the function of all known RTKs. Indeed, human oncogenic Ras and Raf-1 can function in Drosophila, causing phenotypes similar to those seen with RTK gainof-function mutations (Brand and Perrimon, 1994; Casanova et al., 1994). In addition, Raf-1 can rescue a subset of Draf loss-of-function phenotypes and activated Draf can readily phosphorylate human MEK in vitro (W.X Li, unpublished observations). Sos. Sos is a key link from RTK to Ras. In a dosage-sensitive genetic screen, Sos, together with Ras1, was among the first molecules identified as performing crucial early steps in signaling by the Sevenless RTK (Simon et al., 1991). Genetic studies showed Sos is also essential in mediating EGFR and Torso signaling (Rogge et al., 1991; Lu et al., 1993a). Its function as a direct Ras activator was initially suggested by its sequence similarity to yeast CDC25, a guanine nucleotide exchange factor (GEF) bound to Ras (Jones et al., 1991; Rogge et al., 1991; Simon et al., 1991). Further genetic and biochemical studies demonstrated that Sos is indeed an activator of Ras1 and functions to convert GDP-bound Ras1 to the GTP-bound, or active, form in response to RTK activation (Bonfini et al., 1992; Olivier et al., 1993; Simon et al., 1993). Ras. There are two Ras homologs in the fly genome: Ras1 (also known as Ras85D due to its cytogenetic location) and Ras2 (Ras64B). Ras1 is 75% identical to the oncogenic mammalian Ki/Ha ras (p21 ras ) and is considered the only Ras homolog in the fly genome that functions in the Ras-ERK signaling cassette. Ras2, on the other hand, is homologous to mammalian R-ras and does not activate Draf (Fortini et al., 1992; Lu et al., 1993a). The function of Ras1 in mediating Drosophila RTK signaling was first suggested by results of genetic screens for dosage-sensitive modifiers of RTK variants (Simon et al., 1991; Doyle and Bishop, 1993) and was later supported by genetic and biochemical analyses (Lu et al., 1993a; Hou et al., 1995). Embryos devoid of Ras1 function, produced by the germline clone technique, almost completely abolish Torso signaling, as evidenced by phenotypes identical to those of torso loss-of-function mutations in the vast majority (approximately 80%) of these embryos. However, residual tll expression and posterior structures can be detected in the rest of approximately 20% of Ras1 mutant embryos. Raf. Draf is the sole Raf homolog in the fly genome and is 61% and 56% homologous to mammalian B-Raf and Raf-1 (C-Raf), respectively. Draf was first isolated as a lethal mutation, l(1)ph, whose maternal function is essential for the proper determination of terminal cell fates (Perrimon et al., 1985; Ambrosio et al., 1989a). Draf plays a central role in Torso signal transduction, because embryos lacking Draf gene activity have phenotypes identical to those of torso null embryos, resulting in the complete absence of posterior tll expression; weaker Draf

7 662 LI Gene/protein Ligand production torso-like TABLE 1. Molecules Involved in Torso Signaling Molecule/motif Vertebrate homolog Effects of mutation on Torso signaling Reference Secreted; Glycoprotein None Loss of termini Stevens et al., 1990; Savant-Bhonsale et al., 1993 fs(1)nasrat Cell surface molecule None Loss of termini Jiménez et al., 2002 fs(1) ph Cell surface molecule None Loss of termini Jiménez et al., 2002 trunk Cystine knot motif; None Loss of termini Casanova et al., 1995 Sereted; Ligand Receptor Torso Adaptors Drk Dshc Dos Receptor Tyrosine Kinase SH2-SH3 adaptor protein Phosphotyrosine interaction, SH2, PH-like domain pleckstrin homology domain protein Type III RTK GRB2 SHC Gab Loss of termini Sprenger et al., 1989; Casanova and Struhl, 1989 Partial loss of termini Partial loss of termini Partial loss of termini Simon et al., 1991; Hou et al., 1995 Luschnig et al., 2000 Raabe et al., 1996; Herbst et al., 1996 Enzymes Corkscrew SH2 Protein-tyrosine SH-PTP2 Suppresses torso Perkins et al., 1992 phosphatase or SHP2 GOF phenotype Sos Guanine nucleotide exchange factor SOS Partial loss of termini Simon et al., 1991; Hou et al., 1995 Src64B Src family tyrosine kinase Src Reduced Draf activity F. Xia & W.X. Li, unpubl. results PLC Phospholipase PLC NA Cleghon et al., 1996 Core signaling cassette Ras1 Small GTPase Ras Loss of termini Simon et al., 1991; Hou et al., 1995; Lu et al., 1993 Draf or l(1)ph Raf serine/threonine Raf Loss of termini Ambrosio et al., 1989 kinase Dsor1 MAP kinase kinase; dual specificity kinase MEK Loss of termini Tsuda et al., 1993 rolled MAP kinase; serine/ threonine kinase ERK ERK Suppresses torso GOF phenotype Cofactors/scaffolds Ksr Serine/threonine kinase CNK PDZ, SAM, and PH CNK Loss of Ras domain protein signaling Phosphoserine Loss of posterior binding protein midgut Hsp83 Heat shock protein Hsp90 Suppresses torso GOF phenotype Negative regulators D-RasGap Gap1 Sprouty Capicua Ras GTPase activating protein Ras GTPase activating protein FAD/NAD(P)-binding domain protein HMG-box DNAbinding factor Brunner et al., 1994; Li and Li, 2003 KSR Loss of termini Therrien et al., 1995 p120 Ras- GAP Gap1 Sprouty1/2 Cic Increased tll expression Increased tll expression Suppresses weak torso allele Causes torso GOF phenotype Therrien et al., 1998 Li et al., 1997; Kockel et al., 1997 Li and Li, 2003; van der Straten et al., 1997 Cleghon et al., 1998 Hou et al., 1995 Casci et al., 1999 Jiménez et al., 2000 Continues on following page.

8 DROSOPHILA TORSO SIGNALING 663 TABLE 1. (Continued) Gene/protein Groucho Hrs Target genes and protein tailless huckebein Bicoid orthodenticle Other STAT92E Molecule/motif WD-repeat transcrioption corepressor HGF-regulated tyrosine kinase substrate Nuclear hormone receptor Zinc finger transcription factor Homeo domain transcription factor Homeo domain transcription factor SH2-containing transcription factor Vertebrate homolog TLE Hrs Effects of mutation on Torso signaling Increased tll expression Increased tll expression Reference Paroush et al., 1997 Lloyd et al., 2002 Tlx Loss of poserior termini Pignoni et al., 1990 Sp1 Loss of posterior Weigel et al., 1990 midgut Pitx 1/2 Loss of head Driever and Nusslein-Volhard, 1988 Otx Head defects Finkelstein and Perrimon, 1990 STAT5 suppresses torso GOF phenotype Li et al., 2002 a NA, not analyzed; GOF, gain-of-function; HGF, hepatocyte growth factor. alleles exhibit reduced levels of tll expression (Ambrosio et al., 1989b; Melnick et al., 1993; Sprenger et al., 1993). MEK. Genetic screens for mutations that suppress the lethality of a hypomorphic allele of Draf identified mutations in Downstream of Raf1 (Dsor1; Tsuda et al., 1993; Lu et al., 1994). Dsor1 turned out to encode a Drosophila MEK1/2 homolog and the Dsor1 mutations isolated in the above screens were activating mutations that caused constitutive activity of the kinase (Tsuda et al., 1993; Lu et al., 1994). Loss of function Dsor1 mutations reverted the suppression, and these null mutations produced torso loss-of-function phenotypes, demonstrating that Dsor1 mediates Torso signaling and functions downstream of Draf (Tsuda et al., 1993; Lu et al., 1994). ERK. The Drosophila ERK homolog is encoded by the rolled (rl) gene (Biggs and Zipursky, 1992; Biggs et al., 1994; Brunner et al., 1994). Mutant alleles of rl have been isolated repeatedly in genetic screens for genes required for signaling by activated forms of Ras1, Draf, or Torso (Dickson et al., 1996; Karim et al., 1996; Li et al., 2000; Therrien et al., 2000; Li and Li, 2003). A dominant mutation in rolled that resulted in a partially constitutive activation of Rolled/ERK was isolated in a genetic screen for dominant mutations that enable Sevenless signaling in the absence of activation of the Sev RTK (Brunner et al., 1994). This mutation also causes embryonic defects similar to those of torso GOF mutations and is suppressible by tll mutations (Brunner et al., 1994), suggesting that it is required for transducing Ras1 and Draf signals in general. Cofactors and Scaffold Proteins Genetic screens in Drosophila designed to isolate genes essential for Draf activation have identified several genes that encode important modulators such as scaffold proteins, chaperones, and cofactors. These include Kinase Suppressor of Ras (KSR), Connector enhancer of KSR (CNK), Hsp83, and , to name just a few (Therrien et al., 1995; Chang and Rubin, 1997; Kockel et al., 1997; Li et al., 1997; van der Straten et al., 1997; Therrien et al., 1998). The identification of these modulators underscores the complexity in the propagation of signals through the Ras-ERK signaling cassette, as well as the importance of protein protein interactions (see reviews by Morrison, 2001; Dhillon and Kolch, 2002; Chong et al., 2003; Morrison and Davis, 2003). HOW DOES TORSO TRANSDUCE SIGNALS? A combination of genetic and biochemical studies have suggested that Torso signaling shares common features with signaling of other RTKs but also exhibits unique properties. As with all RTKs, Torso presumably dimerizes after binding to extracellular ligands. This presumption is consistent with the finding that all three torso GOF mutations are due to point mutations in the extracellular or transmembrane domains, presumably causing ligandindependent dimerization (Sprenger and Nusslein-Volhard, 1992). Dimerization of the Torso receptor causes its autophosphorylation on multiple tyrosine residues. These phosphotyrosine residues increase its tyrosine kinase activity or serve as specific docking sites for proteins with SH2 domains. Autophosphorylation on tyrosine residues appears to be an essential

9 664 LI step in initiating the signal transduction cascade after RTK activation. When purified Torso protein is allowed to autophosphorylate in vitro, only tyrosine phosphorylation can be detected, and two tyrosine residues (Y630 and Y918) contribute to most of the detectable phosphorylation (Cleghon et al., 1996). When mutant Torso with phenylalanine substitutions of these residues were analyzed in vivo, it was found that py630 plays a positive role, whereas py918 plays a negative role in mediating Torso signals, suggesting that Torso activity is modulated by compensatory mechanisms (Cleghon et al., 1996). Phosphorylation of Y630 provides a docking site for the tyrosine phosphatase Csw, an SHP2 homolog (Perkins et al., 1992; Cleghon et al., 1996), and the negative site py918 serves to recruit the Ras GTPase-activating protein (RasGAP), which negatively regulates Ras activity (Cleghon et al., 1996, 1998; Feldmann et al., 1999). Thus, the strength of the Torso signal is modulated by opposing actions of Csw and RasGAP, contributing to precise control of cell fate specification and pattern formation. Because many vertebrate RTKs bind to both RasGAP and Csw/SHP2, this modulation may be a conserved regulatory mechanism. The SH2-containing phosphotyrosine phosphatase SHP2 in vertebrates can associate with several RTKs and plays a positive role in transducing RTK signals (Lechleider et al., 1993; Tang et al., 1995). However, the precise molecular mechanisms of Csw/SHP function in transducing RTK signaling remain unclear, as several substrates of Csw/SHP2 have been reported. These substrates include RTK itself (Cleghon et al., 1998; Agazie et al., 2003), the adaptor protein Gab/DOS (Herbst et al., 1996; Raabe et al., 1996), the C-terminal Src Kinase (Csk) regulator PAG/Cbp (Zhang et al., 2004), and a negative RTK regulator Sprouty (Spry; Hanafusa et al., 2004). Csw specifically dephosphorylates py918 of Torso, a negative py site capable of binding RasGAP (Cleghon et al., 1998). Similarly, dephosphorylation of Y992 of EGFR by SHP2 prevents the recruitment of RasGAP to the receptor, thus increasing the half-life of activated Ras (Agazie et al., 2003). In a separate report, it has been shown that SHP2 may dephosphorylate a conserved RTK inhibitor Spry, preventing its inhibitory association with Grb2 (Hanafusa et al., 2004). The phosphatase activity of SHP2, therefore, appears essential for its role as a positive mediator of RTK signaling. Deletion of the catalytic site in the phosphatase domain in Xenopus SHP2 results in a dominant-negative mutant (Tang et al., 1995). Similarly, expression of a catalytically dead Csw resulted in dominant-negative phenotypes during Drosophila embryogenesis (M. Melnick and L. Perkins, personal communication). Mutations in human SHP2 that cause Noonan syndrome are associated with significantly increased basal phosphatase activity (Fragale et al., 2004). Aside from dephosphorylating a substrate, Csw/SHP2 may serve as an adaptor protein for recruiting downstream signaling molecules to the RTK. Indeed, it has been shown that after binding to Torso, Csw is phosphorylated by Torso on Y666, which subsequently can be bound by Drk, a Growth factor receptor bound-2 (Grb2) homolog (Cleghon et al., 1998). Ras activation by many RTKs is associated with the recruitment of a Grb2/Drk-Sos complex by means of an autophosphorylated pyxnx motif (Songyang et al., 1993). However, Torso lacks the consensus Drk-binding motif YXNX and does not appear to bind directly to Drk (Cleghon et al., 1996). Phosphorylation of CSW on Y666 effectively creates a Drk-binding site pytni, allowing recruitment of the Drk-SOS complex to Torso, which leads to Ras activation (Cleghon et al., 1998). Because mutation of the major positive phosphotyrosine py630 causes a moderate decrease but does not completely eliminate Torso signaling, efforts were made to identify additional minor py sites. Four additional tyrosine residues (Y644, Y698, Y767, and Y772) were identified and analyzed by mutagenesis and transgenic analysis (Gayko et al., 1999). Taken together with analyses of the two major py sites, it became clear that, upon activation, Torso is autophosphorylated on multiple tyrosine residues located in both the catalytic domain (activation loop) and noncatalytic regions (the kinase insert and C-terminal tail). Analysis of transgenic Torso molecules with tyrosine to phenylalanine substitutions demonstrates that the activation loop tyrosines (Y767 and Y772) are essential for Torso enzymatic activity (Gayko et al., 1999). The C-terminal tail tyrosine (Y918) negatively regulates Torso activity (Cleghon et al., 1996; Gayko et al., 1999). In the kinase insert region of Torso, any single mutation of the four tyrosines (Y630, Y644, Y656, and Y698) has little or no effect on Torso signaling, while simultaneously eliminating all four tyrosine residues (quadruple mutant) completely abolishes Torso signaling (Gayko et al., 1999). These results indicate that multiple tyrosine residues outside of the kinase domain play a synergistic role in mediating Torso signaling, and that Torso recruits downstream molecules by means of the pys located in the kinase insert region. Downstream signaling molecules/adaptors other than Csw that bind to these py residues are yet to be identified. Although Csw is the only positive mediator so far identified that directly binds to Torso, null mutations in csw do not completely abolish Torso signaling (Perkins et al., 1996). Indeed, csw mutant embryos exhibit intact Filzkörper and significant expression levels of the target gene tll (Perkins et al., 1992, 1996). Thus, it is likely that other adaptor proteins or signaling mediators are required to transduce Torso signals. Studies of mammalian RTKs have demonstrated the involvement of several SH2-domain containing adaptors that are recruited to pys after RTK activation. In addition to Csw/SHP2 and Drk/Grb2 mentioned above, such adaptors include SHC (Lai et al., 1995; Luschnig et al., 2000), NCK/DOCK (Garrity et al., 1996; Rao and Zipursky, 1998), Crk (reviewed by Feller, 2001), and Grb7 family proteins (reviewed by Han et al., 2001). Of these adaptors, only SHC has been analyzed and shown to play a role in Torso signaling. SHC can be recruited to an activated RTK and lead to Ras activation (Rozakis-Adcock et al., 1992). SHC contains two py-binding motifs, an N- terminal PTB domain and an SH2 domain in the C-terminus. Studies of mammalian SHC have demonstrated that SHC binds to the NPXpY motif of

10 DROSOPHILA TORSO SIGNALING 665 serve as docking sites for Csw and possibly DSHC and other adaptors or SH2- containing signaling molecules, which may include STAT92E. These molecules redundantly or synergistically recruit Sos to the membrane. Sos converts GDP-Ras1 to GTP-Ras1, leading to activation of the Ras1/Draf/Dsor1/Rolled signaling cassette. In addition, Torso is able to activate Draf by means of a Ras1-independent pathway, and Ras1 plays a role in activating an unknown Draf activator, in addition to binding to Draf. Thus, multiple signaling routes originating from Torso can converge on the Draf kinase, leading to the expression of downstream target genes (Fig. 4). Fig. 4. Mechanism of Torso signal transduction. Ligand (Trunk) binding triggers dimerization and autophosphorylation of Torso on multiple tyrosine residues. Phosphotyrosines located in the activation loop of the tyrosine kinase domain are essential for Torso enzymatic activity. Phosphotyrosines located outside of the kinase domain serve to recruit Csw and possibly DSHC and other adaptors or SH2-containing signaling molecules, which may include STAT92E. The adaptor molecules redundantly or synergistically recruit Son of Sevenless (Sos) to the membrane. Sos converts GDP-Ras1 to GTP-Ras1, linking Torso activation to the Ras1/Draf/Dsor1/Rolled signaling cassette. In addition, Torso is able to activate Draf by means of a Ras1-independent pathway (dashed line 1), and Ras1 plays a role in activating an unknown Draf activator (dashed line 2) in addition to binding to Draf. Thus, multiple signaling routes originating from Torso can converge on the Draf kinase, leading to expression of downstream target genes. In the posterior terminal region, Torso signaling induces target gene tailless (tll) and huckebein (hkb) mainly by derepression, counteracting repressor complexes, including Capicua (Cic) and Groucho (Gro) and possibly other proteins that bind to the regulatory regions of tll and hkb. Gro is a conserved transcription repressor that is recruited to specific genes by its DNA-binding partner Cic. After relief of repression, the expression of tll and hkb depends on participation of transcription activators that may or may not require input from Torso signaling. Gene regulation by Torso in the anterior terminal region is more complex (see text for detail) and is not depicted in this schematic drawing. the EGF receptor by means of its PTB domain and is in turn phosphorylated by EGFR on a specific tyrosine, which provides a binding surface for the SH2 domain of Grb2 (Rozakis-Adcock et al., 1992). Although DSHC binds to EGFR by a similar mechanism, it does not contain a Grb2-binding motif and does not interact with Drk (Lai et al., 1995; van der Geer et al., 1995). Genetic analysis of the dshc gene has demonstrated that DSHC is required for signaling by Torso and EGFR but not by the Sevenless RTK (Luschnig et al., 2000). Significantly, double-mutant analysis demonstrates that three adaptors, DOS, DRK, and DSHC, function in parallel to transduce Torso signals. Thus, mutations in each of the adaptor genes only partially affect Torso signaling, but removal of any two of them simultaneously severely reduces Torso signaling, as assayed by posterior terminal structures and tll expression (Luschnig et al., 2000). Although direct interaction with Torso has not been demonstrated for DSHC, these results indicate an intriguing redundancy among adaptor proteins and suggest that RTKs may use different combinations of adaptors to regulate signaling output qualitatively and quantitatively. Based on genetic and biochemical studies of Torso, the following model can be proposed for Torso signal transduction (Fig. 4). Upon ligand-induced dimerization, Torso becomes autophosphorylated on multiple tyrosine residues. Two phosphotyrosines located in the activation loop (Y767 and Y772) are essential for Torso enzymatic activity. Four phosphotyrosines in the kinase insert region and the C-terminal tail OTHER INTRACELLULAR PATHWAYS MEDIATING TORSO SIGNALING Studies of the mammalian PDGF receptors have shown that they are able to associate with a large number of SH2 domain-containing proteins upon ligand stimulation (reviewed by Heldin and Westermark, 1999; Tallquist and Kazlauskas, 2004). In addition to numerous adaptor proteins that lead to activation of the Ras-ERK signaling pathway, many SH2-containing enzymes that lead to activation of distinct intracellular signaling cascades are implicated. These enzymes include phosphatidylinositol 3 -kinase (PI3K), phospholipase C (PLC)-, STAT proteins, SHP2, GAP, and the Src family of tyrosine kinases (reviewed by Heldin and Westermark, 1999; Tallquist and Kazlauskas, 2004). Mouse knockin experiments replacing the endogenous PDGF receptors with molecules in which the intracellular domain has been swapped with another kinase or has specific tyrosine to phenylalanine substitutions (F series) have been carried out, and the results suggest that many of the downstream signaling pathways are essential for mediating the full range of biological functions of the PDGF receptors (reviewed by Tallquist and Kazlauskas, 2004). For instance, when the PI3K binding site was disrupted, severe developmental defects were observed for PDGF R and partial defects for PDGF R, suggesting that PDGF R relies more on PI3K signaling than PDGF R does (Heuchel et