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Journal ArticleDOI

Drosophila RET contains an active tyrosine kinase and elicits neurotrophic activities in mammalian cells

04 Jul 2005-FEBS Letters (Elsevier)-Vol. 579, Iss: 17, pp 3789-3796

TL;DR: Initial biochemical and functional characterization of the dRET protein in cell culture systems indicate significant conservation between the biological effects elicited by the human and Drosophila RET kinases, and suggest functions for dRET in neuronal differentiation in the fly.

AbstractThe RET receptor tyrosine kinase controls kidney organogenesis and development of subpopulations of enteric and sensory neurons in different vertebrate species, including humans, rodents, chicken and zebrafish. RET is activated by binding to a ligand complex formed by a member of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors bound to its cognate GFRα GPI-linked co-receptor. Despite the absence of GDNF or GFRα molecules in the Drosophila genome, a RET orthologue (dRET) has recently been described in this organism and shown to be expressed in subpopulations of cells of the excretory, digestive and nervous systems, thus resembling the expression pattern of RET in vertebrates. In this study, we report on the initial biochemical and functional characterization of the dRET protein in cell culture systems. Full-length dRET could be produced in mammalian and insect cells. Similar to its human counterpart (hRET), overexpression of dRET resulted in its ligand-independent tyrosine phosphorylation, indicating that it bears an active tyrosine kinase. Unlike hRET, however, the extracellular domain of dRET was unable to interact with mammalian GDNF and GFRα1. Self association between dRET molecules could neither be detected, indicating that dRET is incapable of mediating cell adhesion by homophilic interactions. A chimeric molecule comprising the extracellular domain of hRET and the kinase domain of dRET was constructed and used to probe ligand-mediated downstream activities of the dRET kinase in PC12 cells. GDNF stimulation of cells transfected with the hRET/dRET chimera resulted in neurite outgrowth comparable to that obtained after transfection of wild-type hRET. These results indicate significant conservation between the biological effects elicited by the human and Drosophila RET kinases, and suggest functions for dRET in neuronal differentiation in the fly.

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Citations
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Journal ArticleDOI
TL;DR: Successful improvement of dystrophy independent of hormonal, growth, or immunosuppressive effects is demonstrated, indicating VBP15 merits clinical investigation for DMD and would benefit other chronic inflammatory diseases.
Abstract: Absence of dystrophin makes skeletal muscle more susceptible to injury, resulting in breaches of the plasma membrane and chronic inflammation in Duchenne muscular dystrophy (DMD). Current management by glucocorticoids has unclear molecular benefits and harsh side effects. It is uncertain whether therapies that avoid hormonal stunting of growth and development, and/or immunosuppression, would be more or less beneficial. Here, we discover an oral drug with mechanisms that provide efficacy through anti-inflammatory signaling and membrane-stabilizing pathways, independent of hormonal or immunosuppressive effects. We find VBP15 protects and promotes efficient repair of skeletal muscle cells upon laser injury, in opposition to prednisolone. Potent inhibition of NF-κB is mediated through protein interactions of the glucocorticoid receptor, however VBP15 shows significantly reduced hormonal receptor transcriptional activity. The translation of these drug mechanisms into DMD model mice improves muscle strength, live-imaging and pathology through both preventive and post-onset intervention regimens. These data demonstrate successful improvement of dystrophy independent of hormonal, growth, or immunosuppressive effects, indicating VBP15 merits clinical investigation for DMD and would benefit other chronic inflammatory diseases.

133 citations


Journal ArticleDOI
TL;DR: The discovery of ret opened a field of study that has had a profound impact in cancer research, developmental biology, and neuroscience, and that continues to yield surprises and important insights to this day.
Abstract: The identification of the ret oncogene by Masahide Takahashi and Geoffrey Cooper in 1985 was both serendipitous and paradigmatic ( Takahashi et al. 1985). By transfecting total DNA from a human lymphoma into mouse NIH3T3 cells, they obtained one clone, which in secondary transformants yielded more than 100-fold improvement in transformation efficiency. Subsequent investigations revealed that the ret oncogene was not present as such in the primary lymphoma, but was derived by DNA rearrangement during transfection from normal human sequences of the ret locus. At the time, activation by DNA rearrangement had not been previously described for a transforming gene with the NIH3T3 transfection assay. The discovery of ret opened a field of study that has had a profound impact in cancer research, developmental biology, and neuroscience, and that continues to yield surprises and important insights to this day.

115 citations


Cites background or methods from "Drosophila RET contains an active t..."

  • ...A chimeric approach was used to show that Drosophila RET contains an active tyrosine kinase that is competent to induce neuronal differentiation on activation in PC12 cells (Abrescia et al. 2005)....

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  • ...Drosophila RET is unable to interact with GDNF or GFRa1 of mammalian origin, nor is it capable of mediating cell adhesion (Abrescia et al. 2005)....

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01 Jan 2002
Abstract: The catalytic and signaling activities of RET, a tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF), are controlled by the autophosphorylation of several tyrosine residues in the RET cytoplasmic domain. To analyze the phosphorylation state of individual tyrosines, we generated antibodies recognizing specific phosphotyrosine sites involved in the catalytic (Tyr 905 ) and downstream signaling (Tyr 1015 , Tyr 1062 , and Tyr 1096 ) activities of this receptor. Stimulation with GDNF induced coordinated phosphorylation of the 4 tyrosine residues in neuronal cell lines and in primary cultures of sympathetic neurons isolated from rat superior cervical ganglia. Neurturin and artemin, two other members of the GDNF ligand family, also induced synchronized phosphorylation of RET tyrosines with kinetics comparable to those observed with GDNF. Tyrosine phosphorylation was maximal 15 min after ligand stimulation, decaying thereafter with similar kinetics in all 4 residues. Co-stimulation with a soluble form of the GFR1 co-receptor potentiated ligand-dependent phosphorylation of different intracellular tyrosines to a similar extent and increased the survival of superior cervical ganglion neurons compared with treatment with GDNF alone. In vivo, high levels of phosphorylated Tyr 905 , Tyr 1015 , and Tyr 1062 were detected in embryonic mouse dorsal root ganglia, with a sharp decline at early postnatal stages. Protein transduction of anti-Tyr(P) 1062 antibodies into cultured cells reduced activation of MAPKs ERK1 and ERK2 and the AKT kinase in response to GDNF and diminished GDNF-dependent neuronal differentiation and survival of embryonic sensory neurons from the nodose ganglion. These results demonstrate synchronized utilization of individual RET tyrosine residues in neurons in vivo and reveal

107 citations


Journal ArticleDOI
TL;DR: The presence of RET- and GFR-like genes in insects suggests that a ProtoGFR and a ProtoRET arose early in the evolution of bilaterian animals, but when the ProtoGFL diverged from existing transforming growth factor (TGFβ)-like proteins remains unclear.
Abstract: Four different ligand-receptor binding pairs of the GDNF (glial cell line-derived neurotrophic factor) family exist in mammals, and they all signal via the transmembrane RET receptor tyrosine kinase. In addition, GRAL (GDNF Receptor Alpha-Like) protein of unknown function and Gas1 (growth arrest specific 1) have GDNF family receptor (GFR)-like domains. Orthologs of the four GFRα receptors, GRAL and Gas1 are present in all vertebrate classes. In contrast, although bony fishes have orthologs of all four GDNF family ligands (GFLs), one of the ligands, neurturin, is absent in clawed frog and another, persephin, is absent in the chicken genome. Frog GFRα2 has selectively evolved possibly to accommodate GDNF as a ligand. The key role of GDNF and its receptor GFRα1 in enteric nervous system development is conserved from zebrafish to humans. The role of neurturin, signaling via GFRα2, for parasympathetic neuron development is conserved between chicken and mice. The role of artemin and persephin that signal via GFRα3 and GFRα4, respectively, is unknown in non-mammals. The presence of RET- and GFR-like genes in insects suggests that a ProtoGFR and a ProtoRET arose early in the evolution of bilaterian animals, but when the ProtoGFL diverged from existing transforming growth factor (TGFβ)-like proteins remains unclear. The four GFLs and GFRαs were presumably generated by genome duplications at the origin of vertebrates. Loss of neurturin in frog and persephin in chicken suggests functional redundancy in early tetrapods. Functions of non-mammalian GFLs and prechordate RET and GFR-like proteins remain to be explored.

91 citations


Cites background from "Drosophila RET contains an active t..."

  • ...…kinase domain of D-ret is functional in vitro and can activate many of the same pathways (Ras-ERK, Src, and Jun kinase) as mammalian RET [Read et al., 2005], but, as expected, the less conserved extracellular part of D-ret is unable to interact with mammalian GDNF/GFR 1 [Abrescia et al., 2005]....

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  • ..., 2005], but, as expected, the less conserved extracellular part of D-ret is unable to interact with mammalian GDNF/GFR 1 [Abrescia et al., 2005]....

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Journal ArticleDOI
TL;DR: It is reported that a signaling active version of Ret (RetMEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants, providing a novel mechanism underlying Ret‐mediated cell protection in a situation relevant for human PD.
Abstract: Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (RetMEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of RetMEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD.

59 citations


Cites background from "Drosophila RET contains an active t..."

  • ...The signaling properties and functions of Drosophila Ret are not characterized in great detail, but it is structurally homologous to mammalian Ret and can, to some extent, activate the same signaling pathways (Abrescia et al, 2005)....

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References
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Journal ArticleDOI
06 Dec 2002-Science
TL;DR: The protein kinase complement of the human genome is catalogued using public and proprietary genomic, complementary DNA, and expressed sequence tag sequences to provide a starting point for comprehensive analysis of protein phosphorylation in normal and disease states and a detailed view of the current state of human genome analysis through a focus on one large gene family.
Abstract: We have catalogued the protein kinase complement of the human genome (the "kinome") using public and proprietary genomic, complementary DNA, and expressed sequence tag (EST) sequences. This provides a starting point for comprehensive analysis of protein phosphorylation in normal and disease states, as well as a detailed view of the current state of human genome analysis through a focus on one large gene family. We identify 518 putative protein kinase genes, of which 71 have not previously been reported or described as kinases, and we extend or correct the protein sequences of 56 more kinases. New genes include members of well-studied families as well as previously unidentified families, some of which are conserved in model organisms. Classification and comparison with model organism kinomes identified orthologous groups and highlighted expansions specific to human and other lineages. We also identified 106 protein kinase pseudogenes. Chromosomal mapping revealed several small clusters of kinase genes and revealed that 244 kinases map to disease loci or cancer amplicons.

6,993 citations


"Drosophila RET contains an active t..." refers background in this paper

  • ...actually correspond to pseudogenes and do not encode enzimatically active kinases [14]....

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  • ...structural motifs which unequivocally identify RET as a unique receptor tyrosine kinase in those species [9,13,14]....

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  • ...Fbgn0011829) is an active tyrosine kinase [14], and may therefore exert functions similar to human RET (herein hRET) with regards to downstream signaling....

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Journal ArticleDOI
24 Mar 2000-Science
TL;DR: The nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome is determined using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map.
Abstract: The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

5,958 citations


"Drosophila RET contains an active t..." refers background in this paper

  • ...tified in the Drosophila genome [17,18,29,30], which could be taken to imply that cognate dRET ligands might be ‘‘nonneurotrophic’’ signaling factors....

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Journal ArticleDOI
27 Jan 1994-Nature
TL;DR: It is shown that mice homozygous for a targeted mutation in c-ret develop to term, but die soon after birth, showing renal agenesis or severe dysgenesis, and lacking enteric neurons throughout the digestive tract, indicating an essential component of a signalling pathway required for renal organogenesis and enteric neurogenesis.
Abstract: Receptor tyrosine kinases (RTKs) are cell-surface molecules that transduce signals for cell growth and differentiation. The RTK encoded by the c-ret proto-oncogene is rearranged and constitutively activated in a large proportion of thyroid papillary carcinomas, and germ-line point mutations in c-ret seem to be responsible for the dominantly inherited cancer syndromes multiple endocrine neoplasia (MEN) types 2A and B. The gene is expressed in the developing central and peripheral nervous systems (sensory, autonomic and enteric ganglia) and the excretory system (Wolffian duct and ureteric bud epithelium) of mice, indicating that it may play a role in normal development. Here we show that mice homozygous for a targeted mutation in c-ret develop to term, but die soon after birth, showing renal agenesis or severe dysgenesis, and lacking enteric neurons throughout the digestive tract. Ret is thus an essential component of a signalling pathway required for renal organogenesis and enteric neurogenesis.

1,537 citations


Journal ArticleDOI
24 Mar 2000-Science
TL;DR: The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.
Abstract: A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

1,520 citations


Journal ArticleDOI
28 Jun 1996-Cell
Abstract: We report the expression cloning and characterization of GDNFR-α, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line–derived neurotrophic factor (GDNF). GDNFR-α binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-α with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-α in cells lacking GDNFR-α, and this effect is blocked by a soluble Ret–Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-α, and the Ret PTK is proposed.

1,150 citations