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Title: Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signalingmore » pathway required for determination of cone cell fate in Drosophila. - Highlights: • Knockdown of Cabeza induced rough eye phenotype. • Knockdown of Cabeza induced fusion of cone cells in pupal retinae. • Knockdown of Cabeza induced apoptosis in pupal retinae. • Mutation in EGFR pathway-related genes suppressed the rough eye phenotype. • Cabeza may negatively regulate the EGFR pathway.« less

Authors:
;  [1];  [2];  [3]; ;  [1];  [2]; ; ;  [3];  [4];  [3];  [2];  [1];  [2]
  1. Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan)
  2. (Japan)
  3. Department of Neurology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,Kamigyo-ku, Kyoto 602-8566 (Japan)
  4. North Medical Center, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566 (Japan)
Publication Date:
OSTI Identifier:
22416910
Resource Type:
Journal Article
Resource Relation:
Journal Name: Experimental Cell Research; Journal Volume: 326; Journal Issue: 1; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIBODIES; APOPTOSIS; DROSOPHILA; GENES; GROWTH FACTORS; IN VIVO; MORPHOLOGY; MUTATIONS; NERVOUS SYSTEM DISEASES; PHENOTYPE; RETINA; SIGNALS; SUPEROXIDE DISMUTASE

Citation Formats

Shimamura, Mai, Kyotani, Akane, Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Azuma, Yumiko, Yoshida, Hideki, Binh Nguyen, Thanh, Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Mizuta, Ikuko, Yoshida, Tomokatsu, Mizuno, Toshiki, Nakagawa, Masanori, Tokuda, Takahiko, E-mail: ttokuda@koto.kpu-m.ac.jp, Department of Molecular Pathobiology of Brain Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp, and Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585. Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway. United States: N. p., 2014. Web. doi:10.1016/J.YEXCR.2014.06.004.
Shimamura, Mai, Kyotani, Akane, Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Azuma, Yumiko, Yoshida, Hideki, Binh Nguyen, Thanh, Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Mizuta, Ikuko, Yoshida, Tomokatsu, Mizuno, Toshiki, Nakagawa, Masanori, Tokuda, Takahiko, E-mail: ttokuda@koto.kpu-m.ac.jp, Department of Molecular Pathobiology of Brain Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp, & Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585. Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway. United States. doi:10.1016/J.YEXCR.2014.06.004.
Shimamura, Mai, Kyotani, Akane, Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Azuma, Yumiko, Yoshida, Hideki, Binh Nguyen, Thanh, Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Mizuta, Ikuko, Yoshida, Tomokatsu, Mizuno, Toshiki, Nakagawa, Masanori, Tokuda, Takahiko, E-mail: ttokuda@koto.kpu-m.ac.jp, Department of Molecular Pathobiology of Brain Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp, and Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585. Fri . "Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway". United States. doi:10.1016/J.YEXCR.2014.06.004.
@article{osti_22416910,
title = {Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway},
author = {Shimamura, Mai and Kyotani, Akane and Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 and Azuma, Yumiko and Yoshida, Hideki and Binh Nguyen, Thanh and Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 and Mizuta, Ikuko and Yoshida, Tomokatsu and Mizuno, Toshiki and Nakagawa, Masanori and Tokuda, Takahiko, E-mail: ttokuda@koto.kpu-m.ac.jp and Department of Molecular Pathobiology of Brain Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566 and Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp and Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585},
abstractNote = {Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila. - Highlights: • Knockdown of Cabeza induced rough eye phenotype. • Knockdown of Cabeza induced fusion of cone cells in pupal retinae. • Knockdown of Cabeza induced apoptosis in pupal retinae. • Mutation in EGFR pathway-related genes suppressed the rough eye phenotype. • Cabeza may negatively regulate the EGFR pathway.},
doi = {10.1016/J.YEXCR.2014.06.004},
journal = {Experimental Cell Research},
number = 1,
volume = 326,
place = {United States},
year = {Fri Aug 01 00:00:00 EDT 2014},
month = {Fri Aug 01 00:00:00 EDT 2014}
}
  • Mutations in the proline/tyrosine–nuclear localization signal (PY-NLS) of the Fused in Sarcoma protein (FUS) cause amyotrophic lateral sclerosis (ALS). Here we report the crystal structure of the FUS PY-NLS bound to its nuclear import receptor Karyopherinβ2 (Kapβ2; also known as Transportin). The FUS PY-NLS occupies the structurally invariant C-terminal arch of Kapβ2, tracing a path similar to that of other characterized PY-NLSs. Unlike other PY-NLSs, which generally bind Kapβ2 in fully extended conformations, the FUS peptide is atypical as its central portion forms a 2.5-turn α-helix. The Kapβ2-binding epitopes of the FUS PY-NLS consist of an N-terminal PGKM hydrophobic motif,more » a central arginine-rich α-helix, and a C-terminal PY motif. ALS mutations are found almost exclusively within these epitopes. Each ALS mutation site makes multiple contacts with Kapβ2 and mutations of these residues decrease binding affinities for Kapβ2 (K D for wild-type FUS PY-NLS is 9.5 nM) up to ninefold. Thermodynamic analyses of ALS mutations in the FUS PY-NLS show that the weakening of FUS-Kapβ2 binding affinity, the degree of cytoplasmic mislocalization, and ALS disease severity are correlated.« less
  • Highlights: • RNAi screen identified genetic enhancers for the C. elegans homolog of EAF2. • EAF2 and RBBP4 proteins physically bind to each other and alter transcription. • Overexpression of EAF2 and RBBP4 induces the cell death in prostate cancer cells. - Abstract: The tumor suppressor EAF2 is regulated by androgen signaling and associated with prostate cancer. While EAF2 and its partner ELL have been shown to be members of protein complexes involved in RNA polymerase II transcriptional elongation, the biologic roles for EAF2 especially with regards to the development of cancer remains poorly understood. We have previously identified themore » eaf-1 gene in Caenorhabditiselegans as the ortholog of EAF2, and shown that eaf-1 interacts with the ELL ortholog ell-1 to control development and fertility in worms. To identify genetic pathways that interact with eaf-1, we screened RNAi libraries consisting of transcription factors, phosphatases, and chromatin-modifying factors to identify genes which enhance the effects of eaf-1(tm3976) on fertility. From this screen, we identified lin-53, hmg-1.2, pha-4, ruvb-2 and set-6 as hits. LIN-53 is the C. elegans ortholog of human retinoblastoma binding protein 4/7 (RBBP 4/7), which binds to the retinoblastoma protein and inhibits the Ras signaling pathway. We find that lin-53 showed a synthetic interaction with eaf-1(tm3976) where knockdown of lin-53 in an eaf-1(tm3976) mutant resulted in sterile worms. This phenotype may be due to cell death as the treated worms contain degenerated embryos with increased expression of the ced-1:GFP cell death marker. Further we find that the interaction between eaf-1 and lin-53/RBBP4/7 also exists in vertebrates, which is reflected by the formation of a protein complex between EAF2 and RBBP4/7. Finally, overexpression of either human EAF2 or RBBP4 in LNCaP cells induced the cell death while knockdown of EAF2 in LNCaP enhanced cell proliferation, indicating an important role of EAF2 in controlling the growth and survival of prostate cancer cells. Together these findings identify a novel physical and functional interaction between EAF2 and the Rb pathway.« less
  • Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival ofmore » wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.« less
  • Highlights: {yields} This article described the effects of the EGFR tyrosine kinase inhibitor on the cell proliferation and the apoptosis induction of the colon carcinoma cell lines. {yields} Demonstrated that 326474 is a more potent EGFR inhibitor on colon cancer cells than other three TKIs. {yields} It can be important when considering chemotherapy for colonic cancer patients. -- Abstract: Background: Epidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. Methods: In this study,more » using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. Results: Among the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. Conclusion: Our studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.« less
  • The aspartate aminotransferases (designated GOT1 and GOT2) are two enzymes of Drosophila melanogaster for which naturally occurring electrophoretic variants were not found. There is an electrophoretic difference between D. melanogaster and D. simulans. Since the F/sub 1/ hybrid offspring of these species are sterile, a genetic analysis of the ordinary type cannot be done on differences between the two species. A method was devised to make ''partial hybrids'' in which one chromosome arm is homozygous for melanogaster genes in an otherwise hybrid background. By using this method, Got1 was localized to 2R and Got2 to 2L. Once a gene canmore » be assigned to a chromosome, it may be followed in crossing schemes and mutations from mutagen treatments may be expected. At the locus of Got1 a mutation with low activity was recovered and designated Got1/sup lo/. It was located at a genetic map position of 75 on 2R. A Got2 mutant with a greater migration to the anode was recovered and designated Got2/sup J/. It was located at a genetic map position of 3.0, and in the salivary chromosome was between 22B1 and 22B4 inclusive.« less