TRAF6 is a novel regulator of Notch signaling in Drosophila melanogaster

Notch signaling pathway unravels a fundamental cellular communication system that plays an elemental role in development. It is evident from different studies that the outcome of Notch signaling depends on signal strength, timing, cell type, and cellular context. Since Notch signaling affects a spectrum of cellular activity at various developmental stages by reorganizing itself in more than one way to produce different intensities in the signaling output, it is important to understand the context dependent complexity of Notch signaling and different routes of its regulation. We identified, TRAF6 (Drosophila homolog of mammalian TRAF6) as an interacting partner of Notch intracellular domain (Notch-ICD). TRAF6 genetically interacts with Notch pathway components in trans-heterozygous combinations. Immunocytochemical analysis shows that TRAF6 co-localizes with Notch in Drosophila third instar larval tissues. Our genetic interaction data suggests that the loss-of-function of TRAF6 leads to the rescue of previously identified Kurtz–Deltex mediated wing notching phenotype and enhances Notch protein survival. Co-expression of TRAF6 and Deltex results in depletion of Notch in the larval wing discs and down-regulates Notch targets, Wingless and Cut. Taken together, our results suggest that TRAF6 may function as a negative regulator of Notch signaling.     

p34 is a novel regulator of the oncogenic behavior of NEDD4-1 and PTEN

PTEN is one of the most frequently mutated or deleted tumor suppressors in human cancers. NEDD4-1 was recently identified as the E3 ubiquitin ligase for PTEN; however, a number of important questions remain regarding the role of ubiquitination in regulating PTEN function and the mechanisms by which PTEN ubiquitination is regulated. In the present study, we demonstrated that p34, which was identified as a binding partner of NEDD4-1, controls PTEN ubiquitination by regulating NEDD4-1 protein stability. p34 interacts with the WW1 domain of NEDD4-1, an interaction that enhances NEDD4-1 stability. Expression of p34 promotes PTEN poly-ubiquitination, leading to PTEN protein degradation, whereas p34 knockdown results in PTEN mono-ubiquitination. Notably, an inverse correlation between PTEN and p34/NEDD4-1 levels was confirmed in tumor samples from colon cancer patients. Thus, p34 acts as a key regulator of the oncogenic behavior of NEDD4-1 and PTEN.     

Drosophila Src42A is a negative regulator of RTK signaling

The Src family of nonreceptor tyrosine kinases has been implicated in many signal transduction pathways. However, due to a possible functional redundancy in vertebrates, there is no genetic loss-of-function evidence that any individual Src family member has a crucial role for receptor tyrosine kinase (RTK) signaling. Here we show that an extragenic suppressor of Raf, Su(Raf)1, encodes a Drosophila Src family gene Src42A. Characterization of Src42A mutations shows that Src42A acts independent of Ras1 and that it is, unexpectedly, a negative regulator of RTK signaling. Our study provides the first evidence that Src42A defines a negative regulatory pathway parallel to Ras1 in the RTK signaling cascade. A possible model for Src42A function is discussed.     

The Notch regulator Numb links the Notch and TCR signaling pathways

Both the Notch and TCR signaling pathways play an important role in T cell development, but the links between these signaling pathways are largely unexplored. The adapter protein Numb is a well-characterized inhibitor of Notch and also contains a phosphotyrosine binding domain, suggesting that Numb could provide a link between these pathways. We explored this possibility by investigating the physical interactions among Notch, Numb, and the TCR signaling apparatus and by examining the consequences of a Numb mutation on T cell development. We found that Notch and Numb cocluster with the TCR at the APC contact during Ag-driven T cell-APC interactions in both immature and mature T cells. Furthermore, Numb coimmunoprecipitates with components of the TCR signaling apparatus. Despite this association, T cell development and T cell activation occur normally in the absence of Numb, perhaps due to the expression of the related protein, Numblike. Together our data suggest that Notch and TCR signals may be integrated at the cell membrane, and that Numb may be an important adapter in this process.     

Mouse Crossveinless-2 is the vertebrate homolog of a Drosophila extracellular regulator of BMP signaling

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.     

Mouse Crossveinless-2 is the vertebrate homolog of a Drosophila extracellular regulator of BMP signaling

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.     

The Drosophila melanogaster Suppressor of deltex gene, a regulator of the Notch receptor signaling pathway, is an E3 class ubiquitin ligase.

During development, the Notch receptor regulates many cell fate decisions by a signaling pathway that has been conserved during evolution. One positive regulator of Notch is Deltex, a cytoplasmic, zinc finger domain protein, which binds to the intracellular domain of Notch. Phenotypes resulting from mutations in deltex resemble loss-of-function Notch phenotypes and are suppressed by the mutation Suppressor of deltex [Su(dx)]. Homozygous Su(dx) mutations result in wing-vein phenotypes and interact genetically with Notch pathway genes. We have previously defined Su(dx) genetically as a negative regulator of Notch signaling. Here we present the molecular identification of the Su(dx) gene product. Su(dx) belongs to a family of E3 ubiquitin ligase proteins containing membrane-targeting C2 domains and WW domains that mediate protein-protein interactions through recognition of proline-rich peptide sequences. We have identified a seven-codon deletion in a Su(dx) mutant allele and we show that expression of Su(dx) cDNA rescues Su(dx) mutant phenotypes. Overexpression of Su(dx) also results in ectopic vein differentiation, wing margin loss, and wing growth phenotypes and enhances the phenotypes of loss-of-function mutations in Notch, evidence that supports the conclusion that Su(dx) has a role in the downregulation of Notch signaling.     

The common metabolite glycerol-3-phosphate is a novel regulator of plant defense signaling

Conversion of glycerol to glycerol-3-phosphate (G3P) is one of the highly conserved steps of glycerol metabolism in evolutionary diverse organisms. In plants, G3P is produced either via the glycerol kinase (GK)-mediated phosphorylation of glycerol, or via G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP). We have recently shown that G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Since a mutation in the GLY1-encoded G3Pdh conferred more susceptibility compared to a mutation in the GLI1-encoded GK, we proposed that GLY1 is the major contributor of the total G3P pool that participates in defense against C. higginsianum.Key words: glycerol-3-phosphate, glycerol metabolism, defense, signalingGlycerol and its metabolites are involved in a variety of physiopathological processes in both prokaryotes and eukaryotes, most of which appear to be highly conserved,¹ signifying the fundamental importance of these molecules. Glycerol-3-phosphate (G3P), an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis, participates in the disease-related physiologies of many organisms. In humans, deficiencies in glycerol kinase activity (catalyzing the phosphorylation of glycerol to G3P) result in a variety of metabolic and neurological disorders, while mutations in G3P dehydrogenase (G3Pdh, catalyzing the oxidation of dihydroxyacetone phosphate, DHAP, to G3P) have been linked to sudden infant death syndrome and decreased cardiac Na²⁺ current resulting in ventricular arrhythmias and sudden death.^2,3 Given the fact that glycerol metabolism is conserved between plants and animals, it is conceivable that glycerol and/or G3P might also participate in disease physiology of plants. However, such a role for glycerol and/or G3P remains unexplored.Previous work from our laboratory and others has shown that GLY1-encoded G3Pdh plays an important role in plastidal oleic acid-mediated signaling^4–7 and systemic acquired resistance.⁸ This group of enzymes also plays an important role in fungi and it was recently shown that the disruption of a G3Pdh gene in Colletotrichum gloeosporioides eliminated the ability of the mutant fungus to grow on most carbon sources in vitro, including amino acids and glucose.⁹ However, the G3Pdh knockout (KO) fungus grew normally in the presence of glycerol. The G3Pdh KO fungus also developed normally in its plant host (the round-leaved mallow), prompting the suggestion that glycerol, rather than glucose or sucrose, was the primary transferred source of carbon in planta. This was an unexpected finding, but direct analysis of infected host leaves revealed that their glycerol content did decrease by 40% within 48 hours of infection with C. gloeosporioides.⁹ Since the hemibiotroph C. gloeosporioides appears to be able to utilize glycerol for growth and conidiation in planta, it was possible that glycerol metabolism and associated pathways in the host played an important role in the establishment of infections by Colletotrichum fungi. Furthermore, it was possible that the host had evolved to sense these pathogen-mediated changes in glycerol levels and utilize them as signal(s) to initiate defense.We tested these possibilities by characterizing the role of glycerol metabolism in the Arabidopsis—C. higginsianum interaction (Fig. 1). Infection with C. higginsianum reduced the glycerol content while concomitantly increasing the G3P content in Arabidopsis plants.¹⁰ Mutations in G3P-synthesizing genes gly1 (a G3Pdh) and gli1 (a glycerol kinase),¹¹ resulted in enhanced susceptibility to C. higginsianum. The gly1 plants were much more susceptible than the gli1 plants, suggesting that GLY1-encoded G3Pdh played a more important role in basal resistance to C. higginsianum. Conversely, the act1 mutant, which is impaired in the acylation of G3P with oleic acid (18:1) (Fig. 1), was more resistant to the fungus. The phenotypes seen in the infected gly1 and act1 plants correlated with pathogen-induced G3P levels; C. higginsianum inoculation induced ∼2-fold higher accumulation of G3P in the act1 plants, and ∼2-fold lower G3P in the susceptible gly1 plants, as compared to wild-type plants.¹⁰ To test the hypothesis that G3P synthesized via GLY1 entered the plastidial glycerolipid pathway via the ACT1 catalyzed reaction, we generated act1 gly1 plants. The results supported the hypothesis, as act1 gly1 plants were as susceptible to C. higginsianum as gly1 plants.Open in a separate windowFigure 1A condensed scheme of glycerol metabolism in plants. Glycerol is phosphorylated to glycerol-3-phosphate (G3P) by glycerol kinase (GK; GLI1). G3P can also be generated by G3P dehydrogenase (G3Pdh) via the reduction of dihydroxyacetone phosphate (DHAP) in both the cytosol and the plastids (represented by the oval). G3P generated by this reaction can be transported between the cytosol and plastid stroma. In the plastids G3P is acylated with oleic acid (18:1) by the ACT1-encoded G3P acyltransferase. This ACT1-utilized 18:1 is derived from the stearoyl-acyl carrier protein (ACP)-desaturase (SSI2)-catalyzed desaturation of stearic acid (18:0). The 18:1-ACP generated by SSI2 either enters the prokaryotic lipid biosynthetic pathway through acylation of G3P, or is exported out of the plastids as a coenzyme A (CoA)-thioester to enter the eukaryotic lipid biosynthetic pathway. Other abbreviations used are: PA, phosphatidic acid; Lyso-PA, acyl-G3P; PG, phosphatidylglycerol; MGD, monogalactosyldiacylglycerol; DGD, digalactosyl-diacylglycerol; SL, sulfolipid; DAG, diacylglycerol; DHA, dihydroxyacetone; Gl-3-P, glyceraldehyde-3-phosphate; TCA, tricarboxylic acid cycle. Enzymes as abbreviated as: ACT1, G3P acyltransferase; SSI2, stearoyl acyl carrier protein desaturase; GK, glycerol kinase; G3Pdh, G3P dehydrogenase; TPI, triose phosphate isomerase; DHAK, dihydroxyacetone kinase; F1,6-A, fructose 1,6-biphosphate aldolase; PF6P-P, pyrophosphate fructose-6-phosphate phosphotransferase; G6P-I, glucose-6-phosphate isomerase.More supporting evidence for the role of G3P in defense against C. higginsianum was obtained by overexpressing GLY1 in wild-type plants (Fig. 2A). Similar to act1, overexpression of GLY1 led to a ∼2-fold increase in G3P levels after pathogen inoculation, and these plants were also more resistant to C. higginsianum (Fig. 2B–D). Furthermore, plants overexpressing GLY1 or carrying a mutation in ACT1 exhibited enhanced resistance to C. higginsianum in the pad3 mutant background (Fig. 3).¹⁰ The pad3 plants are compromised in camalexin synthesis, and are hypersusceptible to necrotrophic pathogens.Open in a separate windowFigure 2Pathogen response and G3P levels in transgenic lines overexpressing GLY1. (A) Expression of the GLY1 gene in wild-type or 35S-GLY1 transgenic plant. RNA gel blot analysis was performed on ∼7 µg of total RNA. Ethidium bromide staining of rRNA was used as a loading control. (B) Disease symptoms in C. higginsianum-inoculated Col-0, gly1 or 35S-GLY1 plants at 5 dpi. The plants were spray-inoculated with 10⁶ spores/ml of C. higginsianum. (C) Lesion size in spot-inoculated genotypes. The plants were spot-inoculated with water or 10⁶ spores/ml and the lesion size was measured from 20–30 independent leaves at 6 dpi. Statistical significance was determined using Students t-test. Asterisks indicate data that is statistically significant from that of control (Col-0) (p < 0.05). Error bars indicate SD. (D) G3P levels in Col-0 and 35S-GLY1 plants at 0 and 72 h post-inoculation.Open in a separate windowFigure 3Pathogen response in C. higginsianum-inoculated 35S-GLY1 plants in pad3 background. (A) Disease symptoms on Col-0, pad3 or 35S-GLY1 or 35S-GLY1 pad3 plants spot-inoculated with 10⁶ spores/ml of C. higginsianum. The leaves were photographed at 7 dpi. (B) Lesion size in spot-inoculated Col-0, pad3 or 35S-GLY1 or 35S-GLY1 pad3 plants. The lesion size was measured from 20–30 independent leaves at 7 dpi. Asterisks indicate data that is statistically significant from that of control (Col-0) (p < 0.05). Error bars indicate SD.Exogenous glycerol application increased endogenous G3P and significantly enhanced the ability of the host to resist C. higginsianum.¹⁰ Glycerol-triggered synthesis of G3P also caused a decrease in 18:1 levels, which is known to induce defense signaling, resulting in enhanced basal resistance.^4–7 However, the glycerol-triggered increase in G3P precedes the reduction in 18:1 levels and confers resistance even at time points when low 18:1-mediated signaling is not induced, suggesting that the enhanced resistance after glycerol treatment was due to elevated G3P levels and not to the reduction in 18:1.Understanding the precise roles of G3P will require in-depth analysis of real-time alterations in its levels on a cellular level during pathogenesis. This is complicated by the presence of multiple isoforms of G3Pdh that contribute to the total G3P pool, and by the lack of appropriate tools for monitoring precise changes in intracellular G3P. Systematic analysis of various G3Pdh mutants, in combination with each other and with gli1, should yield novel insights into pathway(s) and steps regulating levels of G3P in the cell.     

Notch signaling: Fringe really is a glycosyltransferase

Fringe modifies the ligand-selectivity of Notch in ways that are crucial for a number of Notch's developmental functions. Recent results have confirmed the suspicion that Fringe is a glycosyltransferase that works in the Golgi complex by modifying Notch's glycosylation state.     

Drosophila Gp150 is required for early ommatidial development through modulation of Notch signaling

Cellular signaling activities must be tightly regulated for proper cell fate control and tissue morphogenesis. Here we report that the Drosophila leucine-rich repeat transmembrane glycoprotein Gp150 is required for viability, fertility and development of the eye, wing and sensory organs. In the eye, Gp150 plays a critical role in regulating early ommatidial formation. Gp150 is highly expressed in cells of the morphogenetic furrow (MF) region, where it accumulates exclusively in intracellular vesicles in an endocytosis-independent manner. Loss of gp150 function causes defects in the refinement of photoreceptor R8 cells and recruitment of other cells, which leads to the formation of aberrant ommatidia. Genetic analyses suggest that Gp150 functions to modulate Notch signaling. Consistent with this notion, Gp150 is co-localized with Delta in intracellular vesicles in cells within the MF region and loss of gp150 function causes accumulation of intracellular Delta protein. Therefore, Gp150 might function in intracellular vesicles to modulate Delta-Notch signaling for cell fate control and tissue morphogenesis.     

Notch signaling activates Yorkie non-cell autonomously in Drosophila

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. Here, we show that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. Our data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth.     

Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling

Although prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.     

Notch signaling and Notch signaling modifiers

Originally discovered nearly a century ago, the Notch signaling pathway is critical for virtually all developmental programs and modulates an astounding variety of pathogenic processes. The DSL (Delta, Serrate, LAG-2 family) proteins have long been considered canonical activators of the core Notch pathway. More recently, a wide and expanding network of non-canonical extracellular factors has also been shown to modulate Notch signaling, conferring newly appreciated complexity to this evolutionarily conserved signal transduction system. Here, I review current concepts in Notch signaling, with a focus on work from the last decade elucidating novel extracellular proteins that up- or down-regulate signal potency.