In Drosophila melanogaster, specification of wing vein cells and sensory organ precursor (SOP) cells, which later give rise to a bristle, requires EGFR signaling. This study shows that Pumilio (Pum), an RNA-binding translational repressor, negatively regulates EGFR signaling in wing vein and bristle development. Loss of Pum function yielded extra wing veins and additional bristles. Conversely, overexpression of Pum eliminated wing veins and bristles. Heterozygotes for Pum produced no phenotype on their own, but greatly enhanced phenotypes caused by the enhancement of EGFR signaling. Conversely, over-expression of Pum suppressed the effects of ectopic EGFR signaling. Components of the EGFR signaling pathway are encoded by mRNAs that have Nanos Response Element (NRE)-like sequences in their 3'UTRs; NREs are known to bind Pum to confer regulation in other mRNAs. This study shows that these NRE-like sequences bind Pum and confer repression on a luciferase reporter in heterologous cells. Taken together, the evidence suggests that Pum functions as a negative regulator of EGFR signaling by directly targeting components of the pathway in Drosophila (Kim, 2012).
In the absence of Pum, extra bristles and wing veins develop, while over-expression of Pum eliminates bristles and wing veins. Several lines of evidence show that the role of Pum is to negatively regulate development of wing veins and bristles. First, loss- and gain-of Pum function produced aberrant wing vein and bristle phenotypes that are inverse to those produced by altered EGFR signaling. Second, reduction of Pum activity greatly enhanced phenotypes associated with reduced EGFR signaling. Third, concomitant expression of Pum suppressed phenotypes associated with ectopic EGFR signaling. In support of the genetic conclusion, it was shown that Pum binds the NRE-like sequence of EGFR, Rl, Sos, and Drk mRNAs and represses translation of a reporter containing these sequences in heterologous cells, suggesting that Pum is a negative regulator of EGFR signaling (Kim, 2012).
To define Pum's role in the development of wing veins and bristles precisely, attempts were made to locate Pum protein and measure Pum activity through a GFP-NRE construct in the 3rd-instar larval and pupal wing imaginal discs where wing vein and SOP cells are specified. A low- level ubiquitous expression of Pum and broad Pum activity was obtained, suggesting that Pum might function as general attenuator of EGFR signaling (Kim, 2012).
This discovery of negative regulation of EGFR signaling by Pum is not confined to Drosophila somatic cells, since it has also been reported in germline cells of C. elegans, cultured human stem cells, and yeast cells. Thus, it is likely that Pum regulation of EGFR signaling is universal and involves diverse developmental contexts, ranging from C. elegans to Drosophila and humans (Kim, 2012).
drk (downstream of receptor kinases) encodes a widely expressed protein with a single SH2 domain and two flanking SH3 domains, homologous to the Sem-5 protein of C. elegans and mammalian GRB2. Genetic analysis suggests that drk function is essential for signaling by the Sevenless receptor tyrosine kinase. DRK biological activity correlates with binding of its SH2 domain to activated receptor tyrosine kinases and concomitant localization of DRK to the plasma membrane. In vitro, DRK also binds directly to the C-terminal tail of SOS, a RAS guanine nucleotide-releasing protein (GNRP), which, like RAS1 and DRK, is required for Sevenless signaling. DRK appears to bind autophosphorylated receptor tyrosine kinases with its SH2 domain and the SOS GNRP through its SH3 domains, thereby coupling receptor tyrosine kinases to RAS activation. The conservation of these signaling proteins during evolution indicates that this is a general mechanism for linking tyrosine kinases to RAS (Olivier, 1993). drk, required for proper signaling by Sevenless, encodes a protein of the structure SH3-SH2-SH3. DRK protein is required for activation of p21Ras1 but not for any subsequent events. DRK protein can bind in vitro to Sevenless and to Son of sevenless (SOS), a putative guanine nucleotide exchange factor for p21Ras1. These results suggest that DRK acts to stimulate the ability of SOS to catalyze p21Ras1 activation by linking Sevenless and SOS in a signaling complex (Oliver, 1993).
Activation of the sevenless protein-tyrosine kinase is required for the proper specification of R7 photoreceptors in the Drosophila eye. The activation of a Ras protein, p21Ras1, is a crucial early event in the signaling pathway, and constitutive activation of p21Ras1 is sufficient to induce all of the effects of sevenless action. Another gene, E(sev)2B, required for proper signaling by sevenless encodes a protein of the structure SH3-SH2-SH3. Evidence is provided that the E(sev)2B protein is required for activation of p21Ras1 but not for any subsequent events, and that this protein can bind in vitro to Sevenless and to Son of sevenless (Sos), a putative guanine nucleotide exchange factor for p21Ras1. These results suggest that the E(sev)2B protein may act to stimulate the ability of Sos to catalyze p21Ras1 activation by linking sevenless and Sos in a signaling complex. The E(sev)2B locus downstream of receptor kinases (drk) (Simon, 1993).
The Drk SH3-SH2-SH3 adaptor protein has been genetically identified in a screen for rate-limiting components acting downstream of the Sevenless (Sev) receptor tyrosine kinase in the developing eye of Drosophila. It provides a link between the activated Sev receptor and Sos, a guanine nucleotide release factor that activates Ras1. A combined biochemical and genetic approach was used to study the interactions between Sev, Drk and Sos. Tyr2546 in the cytoplasmic tail of Sev is required for Drk binding, probably because it provides a recognition site for the Drk SH2 domain. Interestingly, a mutation at this site does not completely block Sev function in vivo. This may suggest that Sev can signal in a Drk-independent, parallel pathway or that Drk can also bind to an intermediate docking protein. Analysis of the Drk-Sos interaction has identified a high affinity binding site for Drk SH3 domains in the Sos tail. The N-terminal Drk SH3 domain is primarily responsible for binding to the tail of Sos in vitro, and for signalling to Ras in vivo (Raabe, 1995).
The Son of sevenless (Sos) protein functions as a guanine nucleotide transfer factor for Ras and interacts with the receptor tyrosine kinase Sevenless through the protein Drk, a homolog of mammalian Grb2. In vivo structure-function analysis reveals that the amino terminus of Sos is essential for its function in flies. A molecule lacking the amino terminus is a potent dominant negative. In contrast, a Sos fragment lacking the Drk binding sites is functional and its activity is dependent on the presence of the Sevenless receptor. Membrane localization of Sos is independent of Drk. This paper discusses a possible role for Drk as an activator of Sos and a Drk-independent interaction between Sos and Sevenless is proposed, one likely to be mediated by the pleckstrin homology domain within the amino terminus (Karlovich, 1995).
Activation of p21ras by receptor tyrosine kinases is thought to result from recruitment of guanine nucleotide exchange factors such as Son-of-sevenless (Sos) to plasma membrane receptor substrates via adaptor proteins such as Grb2. This hypothesis was tested by evaluating the ability of truncation and deletion mutants of Drosophila (d)Sos to enhance [32P]GTP loading of p21ras when expressed in 32P-labeled COS or 293 cells. The dSos catalytic domain (residues 758-1125), expressed without the dSos NH2-terminal (residues 1-757) or adaptor-binding COOH-terminal (residues 1126-1596) regions, exhibits intrinsic exchange activity as evidenced by its rescue of mutant Saccharomyces cerevisiae deficient in endogenous GTP/GDP exchange activity. This dSos catalytic domain fails to affect GTP p21ras levels when expressed in cultured mammalian cells unless the NH2-terminal domain is also present. Surprisingly, the COOH-terminal adaptor binding domain of dSos is not sufficient to confer p21ras exchange activity to the Sos catalytic domain in these cells in the absence of the NH2-terminal domain. This function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin and Dbl homology sequences located just NH2-terminal to the catalytic domain. The results demonstrate a functional role for these pleckstrin and Dbl domains within the dSos protein, and suggest the presence of unidentified cellular elements that interact with these domains and participate in the regulation of p21ras (McCollam, 1995).
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