Torso Receptor Tyrosine Kinase Research Articles

Neither the homeodomain nor the activation domain of Bicoid is specifically required for its down-regulation by the Torso receptor tyrosine kinase cascade.

Bicoid (Bcd) is a maternal morphogen responsible for patterning the head and thorax of the Drosophila embryo. Correct specification of head structure, however, requires the activity of the Torso receptor tyrosine kinase cascade, which also represses expression of Bcd targets at the most anterior tip of the embryo. Here, we investigate the role of both the homeodomain (HD) and the activation domain of Bcd in the anterior repression of its targets. When a Bcd mutant protein whose HD has been replaced by the Gal4 DNA-binding domain is expressed in early embryos, a reporter gene driven by Gal4 DNA-binding sites is first activated in an anterior domain and then repressed from the anterior pole. The down-regulation of Bcd-Gal4 activity requires torso function but does not depend on endogenous bcd activity, indicating that the Bcd protein alone and none of its targets is required to mediate the effect of torso. Functional analysis of a chimeric protein, whose activation domain has been replaced by a generic activation domain, indicates that the activation domain of Bcd is also not specifically required for its down-regulation by Torso. We propose that Torso does not affect the ability of Bcd to bind DNA, but instead directs modification of Bcd or of a potential Bcd co-factor, which renders the Bcd protein unable to activate transcription.

Variation in the number of activated torso receptors correlates with differential gene expression.

Activation of receptor tyrosine kinases triggers many developmental decisions, yet we do not understand how activation of a single receptor can be transduced into different cell responses. The torso pathway in Drosophila provides a model to address this issue since it generates more than one response in the embryo. The torso receptor tyrosine kinase is activated at the embryonic poles under the control of trunk, a protein with similarities to several types of extracellular growth factors. Activation of torso is responsible for the development of a variety of structures, whose appearance can be correlated with activation of at least two different genes along the terminal region. In this study we have analyzed mutations in torso and trunk that express low levels of the respective proteins. We show that different amounts of torso or trunk molecules correlate with the expression of different zygotic genes, implicating changes in the number of activated torso molecules as one of the mechanisms defining differential gene expression. We suggest that variation in the number of activated receptors at the cell surface is a general mechanism that leads to differential gene expression and thus the generation of different cell responses.

Bowel, an odd-skipped homolog, functions in the terminal pathway during Drosophila embryogenesis.

The terminal genes of Drosophila specify non-segmented regions of the larval body that are derived from the anterior and posterior regions of the early embryo. Terminal class genes include both maternal-effect loci (typified by the receptor tyrosine kinase torso) that encode components of a signal transduction cascade and zygotic genes (e.g. tailless and huckebein) that are transcribed at the poles of the embryo in response to the local activation of the pathway. We have characterized a zygotic gene, bowel, that was identified as a zinc finger homolog of the pair-rule segmentation gene odd-skipped. bowel transcripts are initially expressed at both poles of the blastoderm embryo and in a single cephalic stripe. This pattern depends upon torso and tailless activity, but is not affected in huckebein mutants. We isolated and sequenced five mutations that affect the bowel protein, including a nonsense mutation upstream of the zinc fingers and a missense mutation in a putative zinc-chelating residue. bowel mutants die as late embryos with defects in terminal derivatives including the hindgut and proventriculus. Our results indicate that the developmental roles of odd-skipped and bowel have diverged substantially, and that bowel represents a new member of the terminal hierarchy that acts downstream of tailless and mediates a subset of tailless functions in the posterior of the embryo.

Drosophila terminal structure development is regulated by the compensatory activities of positive and negative phosphotyrosine signaling sites on the Torso RTK.

Specification of cell fates in the nonsegmented terminal regions of developing Drosophila embryos is under the control of a signal transduction pathway mediated by the receptor tyrosine kinase Torso (Tor). Here, we identify tyrosines (Y) 630 and 918 as the major sites of Tor autophosphorylation. We demonstrate that mutation of Y630, a site required for association with and tyrosine phosphorylation of the tyrosine phosphatase Corkscrew, decreases the efficiency of Tor signaling. In contrast, mutation of Y918, a site capable of binding mammalian rasGAP and PLC-gammal, increases Tor signaling. Interestingly, when receptors contain mutations in both the Y630 and Y918 sites, Tor signaling is restored to wild-type levels. These results identify a novel mechanism whereby Tor function is regulated using compensatory signals generated from distinct autophosphorylation sites and reveal an underlying signaling pathway for terminal development.

The torso response element binds GAGA and NTF-1/Elf-1, and regulates tailless by relief of repression.

Modulation of transcription factor activity leading to changes in cell behavior (e.g., differentiation versus proliferation) is one of the critical outcomes of receptor tyrosine kinase (RTK) stimulation. In the early Drosophila embryo, activation of the torso (tor) RTK at the poles of the embryo activates a phosphorylation cascade that leads to the spatially specific transcription of the tailless (tll) gene. Our analysis of the tor response element (tor-RE) in the tll promoter indicates that the key activity modulated by the tor RTK pathway is a repressor present throughout the embryo. We have mapped the tor-RE to an 11-bp sequence; using this sequence as the basis for protein purification, we have determined that the proteins GAGA and NTF-1 (also known as Elf-1, product of the grainyhead gene) bind to the tor-RE. We demonstrate that NTF-1 can be phosphorylated by MAPK (mitogen-activated protein kinase), and that tll expression is expanded in embryos lacking maternal NTF-1 activity; these results make NTF-1 a likely target for modulation by the tor RTK pathway in vivo. The data presented here support a model in which activation of the tor RTK at the poles of the embryos leads to inactivation of the repressor and therefore, to transcriptional activation (by activators present throughout the embryo) of the tll gene at the poles of the embryo.

Dissection of the Torso signal transduction pathway in Drosophila

Cell fate choice at the anterior and posterior embryonic termini of the Drosophila embryo requires the activation of a signal transduction pathway regulated by the receptor tyrosine kinase Torso. When Torso, which is uniformly distributed in the egg cell membrane, becomes activated locally at the termini, it triggers a phosphorylation cascade that culminates with localized expression of the transcription factors, tailless and huckebein. Expression of tailless and huckebein in turn determines terminal cell fates. Several genes have been characterized which encode proteins that are involved in Torso signaling: the adaptor protein Drk, the GTP-binding protein Ras1, the guanine nucleotide exchange factor Son of sevenless, and the kinases D-Raf and D-Mek. Genetic and molecular evidence supports a model in which these proteins lie in the same biochemical pathway. When activated by its ligand the membrane-bound receptor tyrosine kinase Torso initiates a signal transduction pathway mediated by Drk, Sos, and Ras1, which in turn activates a phosphorylation cascade mediated by the kinases D-Raf and D-Mek, which ultimately control the localized expression of the transcription factors tailless and huckebein. Recently, we found that D-Raf can be partially activated by Torso in the absence of Ras1, a finding supported by the phenotype of embryos lacking either Drk or Sos activity, as well as by the phenotype of a D-raf mutation that abolishes binding of Ras1 to D-Raf. These findings indicate that full D-Raf activation requires input not only from Ras1 but also from an as yet uncharacterized Ras1-independent pathway. In addition to these molecules we have characterized the putative protein tyrosine phosphatase Corkscrew as a positive transducer downstream of Torso.

The torso receptor tyrosine kinase can activate raf in a ras-independent pathway

Activation of the receptor tyrosine kinase (RTK) torso defines the spatial domains of expression of the transcription factors tailless and huckebein. Previous analyses have demonstrated that Rasl (p21ras) operates upstream of the D-Raf (Raft) serine/threonine kinase in this signaling pathway. By using a recently developed technique of germline mosaics, we find that D-Raf can be activated by torso in the complete absence of Rasl. This result is supported by analysis of D-Raf activation in the absence of either the exchange factor Son of sevenless (Sos) orthe adaptor protein drk (Grb2), as well as by the phenotype of a D-Raf mutation that abolishes binding of Rasl to D-Raf. Our study provides in vivo evidence that Raf can be activated by an RTK in a Ras-independent pathway.

An oncogenic form of human raf can specify terminal body pattern in Drosophila

Terminal portions of the Drosophila body pattern are specified by an extracellular ligand generated at each end of the early syncytial embryo. This ligand triggers the localized transcription of two gap segmentation genes, tailless (tll) and huckebein (hkb) through a signal transduction cascade involving the receptor tyrosine kinase torso (tor) and homologues of ras, raf, and mek (map kinae kinase). In contrast to the ligand, these signal transducing components are expressed ubiquitously. Here, we show that a constitutively active form of human raf1 protein can trigger tll and hkb transcription in Drosophila embryos and specify elements of the terminal body pattern. This result indicates a strong functional conservation between Drosophila and mammalian raf proteins and argues that the localized activity of Drosophila raf (D-raf) normally carries spatial information specifying the end of portions of the body.

Regulation of the dorsal morphogen by the Toll and torso signaling pathways: a receptor tyrosine kinase selectively masks transcriptional repression.

The dorsal (dl) nuclear gradient initiates the differentiation of the mesoderm, neuroectoderm, and dorsal ectoderm by activating and repressing gene expression in the early Drosophila embryo. This gradient is organized by a Toll signaling pathway that shares many common features with the mammalian IL-1 cytokine pathway. Here we present evidence that a second signaling pathway, controlled by the torso (tor) receptor tyrosine kinase, also modulates dl activity. Evidence is presented that the tor 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 tor, such as D-raf and the rolled MAP kinase, mediate this selective block in repression. Normally, the Toll and tor pathways are both active only at the embryonic poles, and consequently, target genes (zen and dpp) that are repressed in middle body regions are expressed at these sites. Constitutive activation of the tor pathway causes severe embryonic defects, including disruptions in gastrulation and mesoderm differentiation, as a result of misregulation of dl target genes. These results suggest that RTK signaling pathways can control gene expression by antirepression, and that multiple pathways can fine-tune the activities of a single transcription factor.

The sevenless signalling cassette mediates Drosophila EGF receptor function during epidermal development

In Drosophila, Drk, an SH2 adaptor protein, Sos, a putative activator of Ras1, Ras1, raf and rolled/MAP kinase have been shown to be required for signalling from the sevenless and the torso receptor tyrosine kinase. From these studies, it was unclear whether these components act in a single linear pathway as suggested by the genetic analysis or whether different components serve to integrate different signals. We have analyzed the effects of removing each of these components during the development of the adult epidermal structures by generating clones of homozygous mutant cells in a heterozygous background. Mutations in each of these signalling components produce a very similar set of phenotypes. These phenotypes resemble those caused by loss-of-function mutations in the Drosophila EGF receptor homolog (DER). It appears that these components form a signalling cassette, which mediates all aspects of DER signalling but that is not required for other signalling processes during epidermal development.

The torso pathway in Drosophila: a model system to study receptor tyrosine kinase signal transduction

In the Drosophila embryo, specification of terminal cell fates that result in the formation of both the head (acron) and tail (telson) regions is under the control of the torso (tor) receptor tyrosine kinase. The current knowledge suggests that activation of tor at the egg pole initiates a signal transduction pathway that is mediated sequentially by the guanine nucleotide releasing factor son of sevenless (Sos), the p21Ras1 GTPase, the serine/threonine kinase D-raf and the tyrosine/threonine kinase MAPKK (Dsor1). Subsequently, it is postulated that activation, possibly by phosphorylation, of a transcription factor at the egg poles activates the transcription of the terminal gap genes tailless and huckebein. These gap genes, which encode putative transcription factors, then control the expression of more downstream factors that ultimately result in head and tail differentiation. Also involved in tor signaling is the non-receptor protein tyrosine phosphatase corkscrew (csw). Here, we review the current model and discuss future research directions in this field.

Down-regulation of the Drosophila morphogen bicoid by the torso receptor-mediated signal transduction cascade

Anterior body pattern in Drosophila is specified by the graded distribution of the bicoid protein (bcd), which activates subordinate genes in distinct anterior domains. Subsequently, transcription of these target genes is repressed at the anterior pole owing to the activity of the receptor tyrosine kinase torso (tor). We show that both activation by bcd and repression by tor can be reproduced by a minimal promoter containing only bcd-binding sites upstream of a naive transcriptional start site. Repression requires the D-raf kinase and is associated with phosphorylation of bcd protein. Repression does not require either tailless or huckebein, which were previously thought to constitute the sole zygotic output of the tor signaling system. Finally, addition of a heterologous transcriptional activation domain to bcd renders the protein insensitive to tormediated repression. We propose that phosphorylation resulting from the activity of the tor signal transduction cascade down-regulates transcriptional activation by the bcd morphogen.

Developmental and molecular characterization of mutations in the Drosophila-raf serine/threonine protein kinase.

Formation of the tail region of the Drosophila larva requires the activities of the terminal class genes. Genetic and molecular analyses of these genes suggests that localized activation of the receptor tyrosine kinase torso at the posterior egg pole triggers a signal transduction pathway. This pathway, mediated through the serine/threonine protein kinase D-raf and the protein tyrosine phosphatase corkscrew, controls the domains of expression of the transcription factors tailless and huckebein. In this paper, we report the molecular and developmental characterization of mutations in the D-raf gene. We show that mutations that alter conserved residues known to be necessary for kinase activity are associated with a null phenotype, demonstrating that D-raf kinase activity is required for its role in torso signaling. Another mutation, D-rafPB26, which prematurely truncates the kinase domain shows a weaker maternal effect phenotype that is strikingly similar to the corkscrew maternal effect phenotype, suggesting that a lower amount of kinase activity decreases the terminal signaling pathway. Finally, molecular and developmental characterization of two mutations that affect the late D-raf zygotic function(s) implies a novel role for D-raf in cell fate establishment in the eye. One of these mutations, D-rafC110, is associated with a single amino acid change within the putative D-raf regulatory region, while the other, D-rafHM-7, most likely reduces the wild-type amount of D-raf protein.

Control of cell fate determination by p21ras/Ras1, an essential component of torso signaling in Drosophila.

Determination of cell fate at the posterior termini of the Drosophila embryo is specified by the activation of the torso (tor) receptor tyrosine kinase. This signaling pathway is mediated by the serine/threonine kinase D-raf and a protein tyrosine phosphatase corkscrew (csw). We found that expression of an activated form of Ras1 during oogenesis resulted in embryos with tor gain-of-function phenotypes. To demonstrate that p21ras/Ras1 mediates tor signaling, we injected mammalian p21ras variants into early Drosophila embryos. We found that the injection of activated p21v-ras rescued the maternal-effect phenotypes of both tor and csw null mutations. These rescuing effects of p21v-ras are dependent on the presence of maternally derived D-raf activity. In addition, wild-type embryos show a terminal-class phenotype resembling csw when injected with p21rasN17, a dominant-negative form of p21ras. Furthermore, we have analyzed the maternal-effect phenotype of Son of sevenless (Sos), a positive regulator of Ras1, and showed that embryos derived from germ cells lacking Sos+ activity exhibit a terminal-class phenotype. Our study demonstrates that 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.

SH2-Containing Phosphotyrosine Phosphatase as a Target of Protein-Tyrosine Kinases

A mouse phosphotyrosine phosphatase containing two Src homology 2 (SH2) domains, Syp, was identified. Syp bound to autophosphorylated epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors through its SH2 domains and was rapidly phosphorylated on tyrosine in PDGF- and EGF-stimulated cells. Furthermore, Syp was constitutively phosphorylated on tyrosine in cells transformed by v-src. This mammalian phosphatase is most closely related, especially in its SH2 domains, to the corkscrew (csw) gene product of Drosophila, which is required for signal transduction downstream of the Torso receptor tyrosine kinase. The Syp gene is widely expressed throughout embryonic mouse development and in adult tissues. Thus, Syp may function in mammalian embryonic development and as a common target of both receptor and nonreceptor tyrosine kinases.

Corkscrew encodes a putative protein tyrosine phosphatase that functions to transduce the terminal signal from the receptor tyrosine kinase torso

We describe the characterization of the Drosophila gene, corkscrew ( csw), which is maternally required for normal determination of cell fates at the termini of the embryo. Determination of terminal cell fates is mediated by a signal transduction pathway that involves a receptor tyrosine kinase, torso, a serine/threonine kinase, D-raf, and the transcription factors, tailless and huckebein. 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 genes. The csw gene encodes a putative nonreceptor protein tyrosine phosphatase covalently linked to two N-terminal SH2 domains, which is similar to the mammalian PTP1C protein.

Reconstruction of the 2.4 Mb human DMD gene by homologous YAC recombination : J.T. Den Dunnen et al. Hum. Mol. Genet. 1, 19–28

Anterior body pattern in Drosophila is specified by the graded distribution of the bicoid protein (bcd), which activates subordinate genes in distinct anterior domains. Subsequently, transcription of these target genes is repressed at the anterior pole owing to the activity of the receptor tyrosine kinase torso (tor). We show that both activation by bcd and repression by tor can be reproduced by a minimal promoter containing only bcd-binding sites upstream of a naive transcriptional start site. Repression requires the D-raf kinase and is associated with phosphorylation of bcd protein. Repression does not require either tailless or huckebein, which were previously thought to constitute the sole zygotic output of the tor signaling system. Finally, addition of a heterologous transcriptional activation domain to bcd renders the protein insensitive to tormediated repression. We propose that phosphorylation resulting from the activity of the tor signal transduction cascade down-regulates transcriptional activation by the bcd morphogen.

Zygotic genes that mediate torso receptor tyrosine kinase functions in the Drosophila melanogaster embryo.

The developmental signal that specifies the fates of cells at the anterior and posterior termini of the Drosophila embryo is transmitted by the torso receptor tyrosine kinase. This paper presents the results of a genetic interaction test for zygotic loci that act downstream of torso in the terminal genetic hierarchy. Tests of 26 zygotic mutants with defects in terminal development indicate that at least 14 reside in this hierarchy. The phenotypes associated with these genes fall into three classes, each of which represents a distinct aspect of terminal development and evolution. Four of the genes have been molecularly cloned and their products include an intercellular communication factor and three kinds of transcription factors.