A Drosophila genetic screen for suppressors of S6kinase-dependent growth identifies the F-box subunit Archipelago/FBXW7

  • Muhammad-Kashif Zahoor
  • Mickael Poidevin
  • Caroline Lecerf
  • Damien Garrido
  • Jacques MontagneEmail author
Original Article


This study was designed to identify novel negative regulators of the Drosophila S6kinase (dS6K). S6K is a downstream effector of the growth-regulatory complex mTORC1 (mechanistic-Target-of-Rapamycin complex 1). Nutrients activate mTORC1, which in turn induces the phosphorylation of S6K to promote cell growth, whereas fasting represses mTORC1 activity. Here, we screened 11,000 RNA-interfering (RNAi) lines and retained those that enhanced a dS6K-dependent growth phenotype. Since RNAi induces gene knockdown, enhanced tissue growth supports the idea that the targeted gene acts as a growth suppressor. To validate the resulting candidate genes, we monitored dS6K phosphorylation and protein levels in double-stranded RNAi-treated S2 cells. We identified novel dS6K negative regulators, including gene products implicated in basal cellular functions, suggesting that feedback inputs modulate mTORC1/dS6K signaling. We also identified Archipelago (Ago), the Drosophila homologue of FBXW7, which is an E3-ubiquitin-ligase subunit that loads ubiquitin units onto target substrates for proteasome-mediated degradation. Despite a previous report showing an interaction between Ago/FBXW7 and dS6K in a yeast two-hybrid assay and the presence of an Ago/FBXW7-consensus motif in the dS6K polypeptide, we could not see a direct interaction in immunoprecipitation assay. Nevertheless, we observed that loss-of-ago/fbxw7 in larvae resulted in an increase in dS6K protein levels, but no change in the levels of phosphorylated dS6K or dS6K transcripts, suggesting that Ago/FBXW7 indirectly controls dS6K translation or stability. Through the identification of novel negative regulators of the downstream target, dS6K, our study may help deciphering the underlying mechanisms driving deregulations of mTORC1, which underlies several human diseases.


Signaling Protein degradation Ago/FBXW7 Genetic screen S2 cells 



We wish to thank the NIG-FLY for the RNAi library and B. Lemaitre, F. Rouyer, F. schweisguth, C. Antonievski for setting an RNAi screening platform; K. Moberg for fly stocks; S. Wardrop for editing the manuscript.

Author contributions

JM conceived the study; MKZ, MP, CL and JM performed experiments; MP and JM analyzed data; DM and JM wrote the paper.


MKZ was supported by a fellowship from the Pakistan Government, DG by fellowships from the French Government (MRT 2011-78) and the Foundation pour la Recherche Médicale (FDT201 4093 0800), JM by Grants from ANR (ANR-05-BLAN-0228-01), Fondation ARC (Projet 1555286), Ligue contre le Cancer (M27218).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflicts of interests.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Supplementary material

438_2018_1529_MOESM1_ESM.pdf (8.8 mb)
Supplementary material 1 (PDF 8972 KB)


  1. Alessi DR, Kozlowski MT, Weng QP, Morrice N, Avruch J (1998) 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr Biol 8:69–81CrossRefGoogle Scholar
  2. Andreazza S, Bouleau S, Martin B, Lamouroux A, Ponien P, Papin C, Chelot E, Jacquet E, Rouyer F (2015) Daytime CLOCK dephosphorylation is controlled by STRIPAK complexes in Drosophila. Cell Rep 11:1266–1279CrossRefGoogle Scholar
  3. Ben-Sahra I, Manning BD (2017) mTORC1 signaling and the metabolic control of cell growth. Curr Opin Cell Biol 45:72–82CrossRefGoogle Scholar
  4. Biondi RM, Kieloch A, Currie RA, Deak M, Alessi DR (2001) The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. Embo J 20:4380–4390CrossRefGoogle Scholar
  5. Biondi RM, Komander D, Thomas CC, Lizcano JM, Deak M, Alessi DR, van Aalten DM (2002) High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site. Embo J 21:4219–4228CrossRefGoogle Scholar
  6. Brini M, Cali T, Ottolini D, Carafoli E (2013) The plasma membrane calcium pump in health and disease. Febs J 280:5385–5397CrossRefGoogle Scholar
  7. Burnett PE, Barrow RK, Cohen NA, Snyder SH, Sabatini DM (1998) RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci USA 95:1432–1437CrossRefGoogle Scholar
  8. Caron A, Richard D, Laplante M (2015) The roles of mTOR complexes in lipid metabolism. Annu Rev Nutr 35:321–348CrossRefGoogle Scholar
  9. Dasgupta B, Yi Y, Chen DY, Weber JD, Gutmann DH (2005) Proteomic analysis reveals hyperactivation of the mammalian target of rapamycin pathway in neurofibromatosis 1-associated human and mouse brain tumors. Cancer Res 65:2755–2760CrossRefGoogle Scholar
  10. Dennis PB, Pullen N, Pearson RB, Kozma SC, Thomas G (1998) Phosphorylation sites in the autoinhibitory domain participate in p70(s6k) activation loop phosphorylation. J Biol Chem 273:14845–14852CrossRefGoogle Scholar
  11. Diaz-Benjumea FJ, Cohen SM (1993) Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila. Cell 75:741–752CrossRefGoogle Scholar
  12. Dibble CC, Manning BD (2013) Signal integration by mTORC1 coordinates nutrient input with biosynthetic output. Nat Cell Biol 15:555–564CrossRefGoogle Scholar
  13. Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, Fellner M, Gasser B, Kinsey K, Oppel S, Scheiblauer S, Couto A, Marra V, Keleman K, Dickson BJ (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448:151–156CrossRefGoogle Scholar
  14. Diggs-Andrews KA, Gutmann DH (2013) Modeling cognitive dysfunction in neurofibromatosis-1. Trends Neurosci 36:237–247CrossRefGoogle Scholar
  15. DiTommaso T, Jones LK, Cottle DL, Program WMG, Gerdin AK, Vancollie VE, Watt FM, Ramirez-Solis R, Bradley A, Steel KP, Sundberg JP, White JK, Smyth IM (2014) Identification of genes important for cutaneous function revealed by a large scale reverse genetic screen in the mouse. PLoS Genet 10:e1004705CrossRefGoogle Scholar
  16. Duval AP, Jeanneret C, Santoro T, Dormond O (2018) mTOR and tumor cachexia. Int J Mol Sci 19:E2225CrossRefGoogle Scholar
  17. Duvel K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, Triantafellow E, Ma Q, Gorski R, Cleaver S, Vander Heiden MG, MacKeigan JP, Finan PM, Clish CB, Murphy LO, Manning BD (2010) Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell 39:171–183CrossRefGoogle Scholar
  18. Finley D (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78:477–513CrossRefGoogle Scholar
  19. Formstecher E, Aresta S, Collura V, Hamburger A, Meil A, Trehin A, Reverdy C, Betin V, Maire S, Brun C, Jacq B, Arpin M, Bellaiche Y, Bellusci S, Benaroch P, Bornens M, Chanet R, Chavrier P, Delattre O, Doye V, Fehon R, Faye G, Galli T, Girault JA, Goud B, de Gunzburg J, Johannes L, Junier MP, Mirouse V, Mukherjee A, Papadopoulo D, Perez F, Plessis A, Rosse C, Saule S, Stoppa-Lyonnet D, Vincent A, White M, Legrain P, Wojcik J, Camonis J, Daviet L (2005) Protein interaction mapping: a Drosophila case study. Genome Res 15:376–384CrossRefGoogle Scholar
  20. Frodin M, Antal TL, Dummler BA, Jensen CJ, Deak M, Gammeltoft S, Biondi RM (2002) A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation. Embo J 21:5396–5407CrossRefGoogle Scholar
  21. Garrido D, Rubin T, Poidevin M, Maroni B, Le Rouzic A, Parvy JP, Montagne J (2015) Fatty acid synthase cooperates with glyoxalase 1 to protect against sugar toxicity. PLoS Genet 11:e1004995CrossRefGoogle Scholar
  22. Goberdhan DC, Ogmundsdottir MH, Kazi S, Reynolds B, Visvalingam SM, Wilson C, Boyd CA (2009) Amino acid sensing and mTOR regulation: inside or out? Biochem Soc Trans 37:248–252CrossRefGoogle Scholar
  23. Groenewoud MJ, Zwartkruis FJ (2013) Rheb and mammalian target of rapamycin in mitochondrial homoeostasis. Open Biol 3:130185CrossRefGoogle Scholar
  24. Guri Y, Nordmann TM, Roszik J (2018) mTOR at the transmitting and receiving ends in tumor immunity. Front Immunol 9:578CrossRefGoogle Scholar
  25. Hase K, Takahashi D, Ebisawa M, Kawano S, Itoh K, Ohno H (2008) Activation-induced cytidine deaminase deficiency causes organ-specific autoimmune disease. PLoS One 3:e3033CrossRefGoogle Scholar
  26. Hennig KM, Neufeld TP (2002) Inhibition of cellular growth and proliferation by dTOR overexpression in Drosophila. Genesis 34:107–110CrossRefGoogle Scholar
  27. Holroyd AK, Michie AM (2018) The role of mTOR-mediated signaling in the regulation of cellular migration. Immunol Lett 196:74–79CrossRefGoogle Scholar
  28. Howell JJ, Ricoult SJ, Ben-Sahra I, Manning BD (2013) A growing role for mTOR in promoting anabolic metabolism. Biochem Soc Trans 41:906–912CrossRefGoogle Scholar
  29. Ibdah JA, Paul H, Zhao Y, Binford S, Salleng K, Cline M, Matern D, Bennett MJ, Rinaldo P, Strauss AW (2001) Lack of mitochondrial trifunctional protein in mice causes neonatal hypoglycemia and sudden death. J Clin Investig 107:1403–1409CrossRefGoogle Scholar
  30. Isotani S, Hara K, Tokunaga C, Inoue H, Avruch J, Yonezawa K (1999) Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro. J Biol Chem 274:34493–34498CrossRefGoogle Scholar
  31. Johannessen CM, Reczek EE, James MF, Brems H, Legius E, Cichowski K (2005) The NF1 tumor suppressor critically regulates TSC2 and mTOR. Proc Natl Acad Sci USA 102:8573–8578CrossRefGoogle Scholar
  32. Johannessen CM, Johnson BW, Williams SM, Chan AW, Reczek EE, Lynch RC, Rioth MJ, McClatchey A, Ryeom S, Cichowski K (2008) TORC1 is essential for NF1-associated malignancies. Curr Biol 18:56–62CrossRefGoogle Scholar
  33. Keshwani MM, von Daake S, Newton AC, Harris TK, Taylor SS (2011) Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1). J Biol Chem 286:23552–23558CrossRefGoogle Scholar
  34. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM (2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110:163–175CrossRefGoogle Scholar
  35. Laplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell 149:274–293CrossRefGoogle Scholar
  36. Lucas X, Ciulli A (2017) Recognition of substrate degrons by E3 ubiquitin ligases and modulation by small-molecule mimicry strategies. Curr Opin Struct Biol 44:101–110CrossRefGoogle Scholar
  37. Magnuson B, Ekim B, Fingar DC (2012) Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem J 441:1–21CrossRefGoogle Scholar
  38. Mao JH, Kim IJ, Wu D, Climent J, Kang HC, DelRosario R, Balmain A (2008) FBXW7 targets mTOR for degradation and cooperates with PTEN in tumor suppression. Science 321:1499–1502CrossRefGoogle Scholar
  39. Moberg KH, Bell DW, Wahrer DC, Haber DA, Hariharan IK (2001) Archipelago regulates cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature 413:311–316CrossRefGoogle Scholar
  40. Moberg KH, Mukherjee A, Veraksa A, Artavanis-Tsakonas S, Hariharan IK (2004) The Drosophila F box protein archipelago regulates dMyc protein levels in vivo. Curr Biol 14:965–974CrossRefGoogle Scholar
  41. Montagne J (2016) A Wacky bridge to mTORC1 dimerization. Dev Cell 36:129–130CrossRefGoogle Scholar
  42. Montagne J, Stewart MJ, Stocker H, Hafen E, Kozma SC, Thomas G (1999) Drosophila S6 kinase: a regulator of cell size. Science 285:2126–2129CrossRefGoogle Scholar
  43. Montagne J, Lecerf C, Parvy JP, Bennion JM, Radimerski T, Ruhf ML, Zilbermann F, Vouilloz N, Stocker H, Hafen E, Kozma SC, Thomas G (2010) The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila. PLoS Genet 6:e1000937CrossRefGoogle Scholar
  44. Moser BA, Dennis PB, Pullen N, Pearson RB, Williamson NA, Wettenhall RE, Kozma SC, Thomas G (1997) Dual requirement for a newly identified phosphorylation site in p70s6k. Mol Cell Biol 17:5648–5655CrossRefGoogle Scholar
  45. O’Connor HF, Huibregtse JM (2017) Enzyme-substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases. Cell Mol Life Sci 74:3363–3375CrossRefGoogle Scholar
  46. Oldham S, Montagne J, Radimerski T, Thomas G, Hafen E (2000) Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev 14:2689–2694CrossRefGoogle Scholar
  47. Perkins LA, Holderbaum L, Tao R, Hu Y, Sopko R, McCall K, Yang-Zhou D, Flockhart I, Binari R, Shim HS, Miller A, Housden A, Foos M, Randkelv S, Kelley C, Namgyal P, Villalta C, Liu LP, Jiang X, Huan-Huan Q, Wang X, Fujiyama A, Toyoda A, Ayers K, Blum A, Czech B, Neumuller R, Yan D, Cavallaro A, Hibbard K, Hall D, Cooley L, Hannon GJ, Lehmann R, Parks A, Mohr SE, Ueda R, Kondo S, Ni JQ, Perrimon N (2015) The Transgenic RNAi project at Harvard Medical School: resources and validation. Genetics 201:843–852CrossRefGoogle Scholar
  48. Philpott C, Tovell H, Frayling IM, Cooper DN, Upadhyaya M (2017) The NF1 somatic mutational landscape in sporadic human cancers. Hum Genom 11:13CrossRefGoogle Scholar
  49. Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, Hemmings BA, Thomas G (1998) Phosphorylation and activation of p70s6k by PDK1. Science 279:707–710CrossRefGoogle Scholar
  50. Radimerski T, Montagne J, Hemmings-Mieszczak M, Thomas G (2002a) Lethality of Drosophila lacking TSC tumor suppressor function rescued by reducing dS6K signaling. Genes Dev 16:2627–2632CrossRefGoogle Scholar
  51. Radimerski T, Montagne J, Rintelen F, Stocker H, van der Kaay J, Downes CP, Hafen E, Thomas G (2002b) dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1. Nat Cell Biol 4:251–255CrossRefGoogle Scholar
  52. Ravid T, Hochstrasser M (2008) Diversity of degradation signals in the ubiquitin-proteasome system. Nat Rev Mol Cell Biol 9:679–690CrossRefGoogle Scholar
  53. Saitoh M, Pullen N, Brennan P, Cantrell D, Dennis PB, Thomas G (2002) Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site. J Biol Chem 277:20104–20112CrossRefGoogle Scholar
  54. Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM (2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 14:1296–1302CrossRefGoogle Scholar
  55. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307:1098–1101CrossRefGoogle Scholar
  56. Saxton RA, Sabatini DM (2017) mTOR signaling in growth, metabolism, and disease. Cell 168:960–976CrossRefGoogle Scholar
  57. Schalm SS, Blenis J (2002) Identification of a conserved motif required for mTOR signaling. Curr Biol 12:632–639CrossRefGoogle Scholar
  58. Schrader EK, Harstad KG, Matouschek A (2009) Targeting proteins for degradation. Nat Chem Biol 5:815–822CrossRefGoogle Scholar
  59. Shima H, Pende M, Chen Y, Fumagalli S, Thomas G, Kozma SC (1998) Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase. Embo J 17:6649–6659CrossRefGoogle Scholar
  60. Shimobayashi M, Hall MN (2014) Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol 15:155–162CrossRefGoogle Scholar
  61. Switon K, Kotulska K, Janusz-Kaminska A, Zmorzynska J, Jaworski J (2017) Molecular neurobiology of mTOR. Neuroscience 341:112–153CrossRefGoogle Scholar
  62. Tahmasebi S, Khoutorsky A, Mathews MB, Sonenberg N (2018) Translation deregulation in human disease. Nat Rev Mol Cell Biol 19:791–807CrossRefGoogle Scholar
  63. The I, Hannigan GE, Cowley GS, Reginald S, Zhong Y, Gusella JF, Hariharan IK, Bernards A (1997) Rescue of a Drosophila NF1 mutant phenotype by protein kinase A. Science 276:791–794CrossRefGoogle Scholar
  64. Welcker M, Clurman BE (2008) FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nat Rev Cancer 8:83–93CrossRefGoogle Scholar
  65. Yeh CH, Bellon M, Nicot C (2018) FBXW7: a critical tumor suppressor of human cancers. Mol Cancer 17:115CrossRefGoogle Scholar
  66. Zhang H, Stallock JP, Ng JC, Reinhard C, Neufeld TP (2000) Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev 14:2712–2724CrossRefGoogle Scholar
  67. Zhang T, Zhou Q, Ogmundsdottir MH, Moller K, Siddaway R, Larue L, Hsing M, Kong SW, Goding CR, Palsson A, Steingrimsson E, Pignoni F (2015) Mitf is a master regulator of the v-ATPase, forming a control module for cellular homeostasis with v-ATPase and TORC1. J Cell Sci 128:2938–2950CrossRefGoogle Scholar
  68. Zheng N, Shabek N (2017) Ubiquitin ligases: structure, function, and regulation. Annu Rev Biochem 86:129–157CrossRefGoogle Scholar
  69. Zimmer S, Stocker A, Sarbolouki MN, Spycher SE, Sassoon J, Azzi A (2000) A novel human tocopherol-associated protein: cloning, in vitro expression, and characterization. J Biol Chem 275:25672–25680CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Muhammad-Kashif Zahoor
    • 1
    • 2
  • Mickael Poidevin
    • 1
  • Caroline Lecerf
    • 1
  • Damien Garrido
    • 1
    • 3
  • Jacques Montagne
    • 1
    Email author
  1. 1.Institute for Integrative Biology of the Cell (I2BC), CNRS, Université Paris-Sud, CEA, UMR9198Gif-sur-YvetteFrance
  2. 2.Department of ZoologyGovernment College UniversityFaisalabadPakistan
  3. 3.IRIC, Université de MontréalMontrealCanada

Personalised recommendations