A Drosophila genetic screen for suppressors of S6kinase-dependent growth identifies the F-box subunit Archipelago/FBXW7
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.
KeywordsSignaling 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.
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.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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