scholarly journals A Genetic Screen for Modifiers of Drosophila Src42A Identifies Mutations in Egfr, rolled and a Novel Signaling Gene

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 697-711
Author(s):  
Qian Zhang ◽  
Qingxia Zheng ◽  
Xiangyi Lu
Keyword(s):  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Photoreceptor Cells ◽  
Mapk Signaling ◽  
Close Relative ◽  
Loss Of Function ◽  
Genetic Screens ◽  
Wing Vein ◽  
Rtk Signaling ◽  
Vein Formation

Abstract Drosophila Src42A, a close relative of the vertebrate c-Src, has been implicated in the Ras-Mapk signaling cascade. An allele of Src42A, Su(Raf)1, dominantly suppresses the lethality of partial loss-of-function Raf mutations. To isolate genes involved in the same pathway where Src42A functions, we carried out genetic screens for dominant suppressor mutations that prevented Su(Raf)1 from suppressing Raf. Thirty-six mutations representing at least five genetic loci were recovered from the second chromosome. These are Drosophila EGF Receptor (Egfr), rolled, Src42A, and two other new loci, one of which was named semang (sag). During embryogenesis, sag affects the development of the head, tail, and tracheal branches, suggesting that it participates in the pathways of Torso and DFGF-R1 receptor tyrosine kinases. sag also disrupts the embryonic peripheral nervous system. During the development of imaginal discs, sag affects two processes known to require Egfr signaling: the recruitment of photoreceptor cells and wing vein formation. Thus sag functions in several receptor tyrosine kinase (RTK)-mediated processes. In addition, sag dominantly enhances the phenotypes associated with loss-of-function Raf and rl, but suppresses those of activated Ras1V12 mutation. This work provides the first genetic evidence that both Src42A and sag are modulators of RTK signaling.

scholarly journals rhomboid and Star interact synergistically to promote EGFR/MAPK signaling during Drosophila wing vein development

Development ◽  
1999 ◽  
Vol 126 (12) ◽  
pp. 2663-2676 ◽  
Author(s):  
A. Guichard ◽  
B. Biehs ◽  
M.A. Sturtevant ◽  
L. Wickline ◽  
J. Chacko ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Mapk Pathway ◽  
Developmental Regulation ◽  
Mapk Signaling ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Wing Vein ◽  
Vein Formation ◽  
Necessary And Sufficient

Genes of the ventrolateral group in Drosophila are dedicated to developmental regulation of Egfr signaling in multiple processes including wing vein development. Among these genes, Egfr encodes the Drosophila EGF-Receptor, spitz (spi) and vein (vn) encode EGF-related ligands, and rhomboid (rho) and Star (S) encode membrane proteins. In this study, we show that rho-mediated hyperactivation of the EGFR/MAPK pathway is required for vein formation throughout late larval and early pupal development. Consistent with this observation, rho activity is necessary and sufficient to activate MAPK in vein primordium during late larval and early pupal stages. Epistasis studies using a dominant negative version of Egfr and a ligand-independent activated form of Egfr suggest that rho acts upstream of the receptor. We show that rho and S function in a common aspect of vein development since loss-of-function clones of rho or S result in nearly identical non-autonomous loss-of-vein phenotypes. Furthermore, mis-expression of rho and S in wild-type and mutant backgrounds reveals that these genes function in a synergistic and co-dependent manner. In contrast, spi does not play an essential role in the wing. These data indicate that rho and S act in concert, but independently of spi, to promote vein development through the EGFR/MAPK signaling pathway.

Differential Activity of Ras1 during Patterning of the Drosophila Dorsoventral Axis

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1545-1557 ◽  
Author(s):  
Jon D Schnorr ◽  
Celeste A Berg
Keyword(s):  
Tyrosine Kinases ◽  
P Element ◽  
Embryonic Patterning ◽  
Loss Of Function ◽  
Wing Vein ◽  
Border Cell ◽  
Specific Effects ◽  
Vein Formation ◽  
Allele Specific ◽  
Development Analysis

In Drosophila, the Ras1 gene is required downstream of receptor tyrosine kinases for correct eye development, embryonic patterning, wing vein formation, and border cell migration. Here we characterize a P-element allele of Ras1, Ras15703, that affects viability, eye morphogenesis, and early and late stages of oogenesis. Flies transheterozgyous for Ras15703 and existing EMS-induced Ras1 alleles are viable and exhibit a range of eye and eggshell defects. Differences in the severity of these phenotypes in different tissues suggest that there are allele-specific effects of Ras1 in development. Analysis of rescue constructs demonstrates that these differential phenotypes are due to loss of function in Ras1 alone and not due to effects on neighboring genes. Females mutant at the Ras1 locus lay eggs with reduced or missing dorsal eggshell structures. We observe dominant interactions between Ras1 mutants and other dorsoventral pathway mutants, including and Egfrtop and gurken. Ras1 is also epistatic to K10. Unlike Egfrtop and gurken mutants, however, Ras1 females are moderately fertile, laying eggs with ventralized eggshells that can hatch normal larvae. These results suggest that Ras1 may have a different requirement in the patterning of the eggshell axis than in the patterning of the embryonic axis during oogenesis.

scholarly journals MKP-3 Has Essential Roles as a Negative Regulator of the Ras/Mitogen-Activated Protein Kinase Pathway during Drosophila Development

2004 ◽  
Vol 24 (2) ◽  
pp. 573-583 ◽  
Author(s):  
Myungjin Kim ◽  
Guang-Ho Cha ◽  
Sunhong Kim ◽  
Jun Hee Lee ◽  
Jeehye Park ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Fate ◽  
Photoreceptor Cells ◽  
Mapk Signaling ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Loss Of Function ◽  
Wing Vein ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein kinase (MAPK) phosphatase 3 (MKP-3) is a well-known negative regulator in the Ras/extracellular signal-regulated kinase (ERK)-MAPK signaling pathway responsible for cell fate determination and proliferation during development. However, the physiological roles of MKP-3 and the mechanism by which MKP-3 regulates Ras/Drosophila ERK (DERK) signaling in vivo have not been determined. Here, we demonstrated that Drosophila MKP-3 (DMKP-3) is critically involved in cell differentiation, proliferation, and gene expression by suppressing the Ras/DERK pathway, specifically binding to DERK via the N-terminal ERK-binding domain of DMKP-3. Overexpression of DMKP-3 reduced the number of photoreceptor cells and inhibited wing vein differentiation. Conversely, DMKP-3 hypomorphic mutants exhibited extra photoreceptor cells and wing veins, and its null mutants showed striking phenotypes, such as embryonic lethality and severe defects in oogenesis. All of these phenotypes were highly similar to those of the gain-of-function mutants of DERK/rl. The functional interaction between DMKP-3 and the Ras/DERK pathway was further confirmed by genetic interactions between DMKP-3 loss-of-function mutants or overexpressing transgenic flies and various mutants of the Ras/DERK pathway. Collectively, these data provide the direct evidences that DMKP-3 is indispensable to the regulation of DERK signaling activity during Drosophila development.

Identification of Genomic Regions That Interact With a Viable Allele of the Drosophila Protein Tyrosine Phosphatase Corkscrew

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 733-748
Author(s):  
Lucy Firth ◽  
Jebecka Manchester ◽  
James A Lorenzen ◽  
Martin Baron ◽  
Lizabeth A Perkins
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Kinases ◽  
Tyrosine Phosphatase ◽  
Current Evidence ◽  
Wing Vein ◽  
Protein Tyrosine ◽  
Rtk Signaling ◽  
Isolation And Characterization ◽  
Drosophila Protein ◽  
Genomic Regions

Abstract Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Previously, Csw has been demonstrated to function throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unambiguously elucidated, current evidence suggests that Csw plays more than one role during transduction of the RTK signal and, further, the molecular mechanism of Csw function differs depending upon the RTK in question. The isolation and characterization of a new, spontaneously arising, viable allele of csw, cswlf, has allowed us to undertake a genetic approach to identify loci required for Csw function. The rough eye and wing vein gap phenotypes exhibited by adult flies homo- or hemizygous for cswlf has provided a sensitized background from which we have screened a collection of second and third chromosome deficiencies to identify 33 intervals that enhance and 21 intervals that suppress these phenotypes. We have identified intervals encoding known positive mediators of RTK signaling, e.g., drk, dos, Egfr, E(Egfr)B56, pnt, Ras1, rolled/MAPK, sina, spen, Src64B, Star, Su(Raf)3C, and vein, as well as known negative mediators of RTK signaling, e.g., aos, ed, net, Src42A, sty, and su(ve). Of particular interest are the 5 lethal enhancing intervals and 14 suppressing intervals for which no candidate genes have been identified.

scholarly journals Control of Growth and Differentiation byDrosophila RasGAP, a Homolog of p120 Ras–GTPase-Activating Protein

1999 ◽  
Vol 19 (3) ◽  
pp. 1928-1937 ◽  
Author(s):  
Pascale Feldmann ◽  
Eva N. Eicher ◽  
Sally J. Leevers ◽  
Ernst Hafen ◽  
David A. Hughes
Keyword(s):  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Intracellular Localization ◽  
Ectopic Expression ◽  
Gtpase Activating Protein ◽  
Wing Vein ◽  
Sh2 Domains ◽  
Ras Gtpase ◽  
Vein Formation

ABSTRACT Mammalian Ras GTPase-activating protein (GAP), p120 Ras-GAP, has been implicated as both a downregulator and effector of Ras proteins, but its precise role in Ras-mediated signal transduction pathways is unclear. To begin a genetic analysis of the role of p120 Ras-GAP we identified a homolog from the fruit flyDrosophila melanogaster through its ability to complement the sterility of a Schizosaccharomyces pombe (fission yeast) gap1 mutant strain. Like its mammalian homolog,Drosophila RasGAP stimulated the intrinsic GTPase activity of normal mammalian H-Ras but not that of the oncogenic Val12 mutant. RasGAP was tyrosine phosphorylated in embryos and its Src homology 2 (SH2) domains could bind in vitro to a small number of tyrosine-phosphorylated proteins expressed at various developmental stages. Ectopic expression of RasGAP in the wing imaginal disc reduced the size of the adult wing by up to 45% and suppressed ectopic wing vein formation caused by expression of activated forms of Breathless and Heartless, two Drosophila receptor tyrosine kinases of the fibroblast growth factor receptor family. The in vivo effects of RasGAP overexpression required intact SH2 domains, indicating that intracellular localization of RasGAP through SH2-phosphotyrosine interactions is important for its activity. These results show that RasGAP can function as an inhibitor of signaling pathways mediated by Ras and receptor tyrosine kinases in vivo. Genetic interactions, however, suggested a Ras-independent role for RasGAP in the regulation of growth. The system described here should enable genetic screens to be performed to identify regulators and effectors of p120 Ras-GAP.

Regulation of Drosophila wing vein patterning: net encodes a bHLH protein repressing rhomboid and is repressed by rhomboid-dependent Egfr signalling

Development ◽  
2000 ◽  
Vol 127 (21) ◽  
pp. 4729-4741 ◽  
Author(s):  
D. Brentrup ◽  
H. Lerch ◽  
H. Jackle ◽  
M. Noll
Keyword(s):  
Imaginal Disc ◽  
Egf Receptor ◽  
Ectopic Expression ◽  
Wing Disc ◽  
Bhlh Protein ◽  
Wing Vein ◽  
Drosophila Wing ◽  
Vein Formation ◽  
Mutual Repression ◽  
Vein Patterning

The stereotyped pattern of veins in the Drosophila wing is generated in response to local EGF signalling. Mutations in the rhomboid (rho) gene, which encodes a sevenpass membrane protein required to enhance signalling transmitted by the EGF receptor (Egfr), inhibit vein development and disrupt the vein pattern. By contrast, net mutations produce ectopic veins in intervein regions. We have cloned the net gene and show that it encodes a basic HLH protein that probably acts as a transcriptional repressor. net and rho are expressed in mutually exclusive patterns during the development of the wing imaginal disc. Lack of net activity causes rho expression to expand, and vice versa. Furthermore, ectopic expression of net or rho results in their mutual repression and thus suppresses vein formation or generates tube-like wings composed of vein-like tissue. Egfr signalling and net exert mutually antagonising activities during the specification of vein versus intervein fate. While Egfr signalling represses net transcription, net exhibits a two-tiered control by repressing rho transcription and interfering with Egfr signalling downstream of Rho. Our results further suggest that net is required to maintain intervein development by restricting Egfr signalling, which promotes vein development, to the Net-free vein regions of the wing disc.

Differential requirement for STAT by gain-of-function and wild-type receptor tyrosine kinase Torso in Drosophila

Development ◽  
2002 ◽  
Vol 129 (18) ◽  
pp. 4241-4248 ◽  
Author(s):  
Willis X. Li ◽  
Herve Agaisse ◽  
Bernard Mathey-Prevot ◽  
Norbert Perrimon
Keyword(s):  
Tyrosine Kinases ◽  
Mapk Signaling ◽  
Wild Type ◽  
Gain Of Function ◽  
Normal Functions ◽  
Rtk Signaling ◽  
Ectopic Gene Expression ◽  
Mutant Forms ◽  
Type Receptor ◽  
Stat Pathway

Malignant transformation frequently involves aberrant signaling from receptor tyrosine kinases (RTKs). These receptors commonly activate Ras/Raf/MEK/MAPK signaling but when overactivated can also induce the JAK/STAT pathway, originally identified as the signaling cascade downstream of cytokine receptors. Inappropriate activation of STAT has been found in many human cancers. However, the contribution of the JAK/STAT pathway in RTK signaling remains unclear. We have investigated the requirement of the JAK/STAT pathway for signaling by wild-type and mutant forms of the RTK Torso (Tor) using a genetic approach in Drosophila. Our results indicate that the JAK/STAT pathway plays little or no role in signaling by wild-type Tor. In contrast, we find that STAT, encoded by marelle (mrl; DStat92E), is essential for the gain-of-function mutant Tor (TorGOF) to activate ectopic gene expression. Our findings indicate that the Ras/Raf/MEK/MAPK signaling pathway is sufficient to mediate the normal functions of wild-type RTK, whereas the effects of gain-of-function mutant RTK additionally require STAT activation.

scholarly journals CRISPRi enables isoform-specific loss-of-function screens and identification of gastric cancer-specific isoform dependencies

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Rebecca Davies ◽  
Ling Liu ◽  
Sheng Taotao ◽  
Natasha Tuano ◽  
Richa Chaturvedi ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Functional Dependencies ◽  
Loss Of Function ◽  
Genetic Screens ◽  
Transcript Isoforms ◽  
Specific Loss ◽  
Current Loss ◽  
Specific Transcript ◽  
Multiple Promoters ◽  
Poor Patient

Abstract Introduction Genes contain multiple promoters that can drive the expression of various transcript isoforms. Although transcript isoforms from the same gene could have diverse and non-overlapping functions, current loss-of-function methodologies are not able to differentiate between isoform-specific phenotypes. Results Here, we show that CRISPR interference (CRISPRi) can be adopted for targeting specific promoters within a gene, enabling isoform-specific loss-of-function genetic screens. We use this strategy to test functional dependencies of 820 transcript isoforms that are gained in gastric cancer (GC). We identify a subset of GC-gained transcript isoform dependencies, and of these, we validate CIT kinase as a novel GC dependency. We further show that some genes express isoforms with opposite functions. Specifically, we find that the tumour suppressor ZFHX3 expresses an isoform that has a paradoxical oncogenic role that correlates with poor patient outcome. Conclusions Our work finds isoform-specific phenotypes that would not be identified using current loss-of-function approaches that are not designed to target specific transcript isoforms.

Drosophila Gain-of-Function Mutant RTK Torso Triggers Ectopic Dpp and STAT Signaling

Genetics ◽  
2003 ◽  
Vol 164 (1) ◽  
pp. 247-258 ◽  
Author(s):  
Jinghong Li ◽  
Willis X Li
Keyword(s):  
Tyrosine Kinases ◽  
Target Genes ◽  
Tor Signaling ◽  
Gain Of Function ◽  
Essential Requirement ◽  
Downstream Target ◽  
Rtk Signaling ◽  
Genome Wide ◽  
A Genome ◽  
Genomic Regions

Abstract Overactivation of receptor tyrosine kinases (RTKs) has been linked to tumorigenesis. To understand how a hyperactivated RTK functions differently from wild-type RTK, we conducted a genome-wide systematic survey for genes that are required for signaling by a gain-of-function mutant Drosophila RTK Torso (Tor). We screened chromosomal deficiencies for suppression of a gain-of-function mutation tor (torGOF), which led to the identification of 26 genomic regions that, when in half dosage, suppressed the defects caused by torGOF. Testing of candidate genes in these regions revealed many genes known to be involved in Tor signaling (such as those encoding the Ras-MAPK cassette, adaptor and structural molecules of RTK signaling, and downstream target genes of Tor), confirming the specificity of this genetic screen. Importantly, this screen also identified components of the TGFβ (Dpp) and JAK/STAT pathways as being required for TorGOF signaling. Specifically, we found that reducing the dosage of thickveins (tkv), Mothers against dpp (Mad), or STAT92E (aka marelle), respectively, suppressed torGOF phenotypes. Furthermore, we demonstrate that in torGOF embryos, dpp is ectopically expressed and thus may contribute to the patterning defects. These results demonstrate an essential requirement of noncanonical signaling pathways for a persistently activated RTK to cause pathological defects in an organism.

scholarly journals Dominant Mutations of Drosophila MAP Kinase Kinase and Their Activities in Drosophila and Yeast MAP Kinase Cascades

Genetics ◽  
1997 ◽  
Vol 146 (1) ◽  
pp. 263-273 ◽  
Author(s):  
Young-Mi Lim ◽  
Leo Tsuda ◽  
Yoshihiro H Inoue ◽  
Kenji Irie ◽  
Takashi Adachi-Yamada ◽  
...  
Keyword(s):  
Map Kinase ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Kinase Domain ◽  
Nucleotide Sequencing ◽  
Gain Of Function ◽  
Highly Sensitive ◽  
Mitogen Activated Protein ◽  
Signal Specificity ◽  
Map Kinase Kinase

Eight alleles of Dsor1 encoding a Drosophila homologue of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D-raf. These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors. They suppressed mutations for receptor tyrosine kinases (RTKs); torso (tor), sevenless (sev) and to a lesser extent Drosophila EGF receptor (DER). Furthermore, the Dsor1 alleles showed no significant interaction with gain-of-function mutations of DER. The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity. Expression of Dsor1 in budding yeast demonstrated that Dsor1 can activate yeast MAP kinase homologues if a proper activator of Dsor1 is coexpressed. Nucleotide sequencing of the Dsor1 mutant genes revealed that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain. The results suggest that they function as suppressors due to increased reactivity to upstream factors.

Sign in / Sign up

Export Citation Format

Share Document