Figure 1. Drosophila TORC Is Activated by Starvation and Oxidative Stress.

(A) Top: schematic of TORC showing the CREB binding domain (CBD), transactivation domain (TAD), nuclear import (NLS) and export (NES) sequences, calcineurin (Cn) recognition motif, and regulatory phosphorylation site (Ser157). Alignment with the mammalian TORC1 sequence and the consensus motif for phosphorylation by salt-inducible kinases (SIKs) and other members of the AMPK family of Ser/Thr kinases is shown. Hydrophobic (Ψ) and basic (B) residues are indicated. Bottom left: immunoblot showing relative amounts of TORC protein in larvae (L1–L3), pupae (P), and adult male flies. Bottom right: qPCR analysis of TORC mRNA amounts at different developmental stages.

(B) Top: immunoblot of Drosophila TORC protein in Drosophila S2 cells exposed to forskolin + isobutylmethylxanthine (F/I), staurosporine (STS), or DMSO vehicle (CON). Amounts of phospho-TORC(Ser157) and total TORC protein recovered from anti-FLAG epitope immunoprecipitates in cells expressing either wild-type (WT) or Ser157Ala mutant Drosophila TORC are shown. Bottom: immunoblot of 14-3-3 proteins recovered from immunoprecipitates of WT or Ser157Ala mutant TORC in HEK293T cells following exposure to forskolin (FSK), STS, or control vehicle. Amounts of phospho-TORC(Ser157) and total TORC shown.

(C) Endogenous Drosophila TORC protein staining by immunofluorescence analysis of Drosophila KC-167 cells under basal conditions (CON) and following exposure to STS. DAPI staining was used to visualize nuclei. Dephosphorylation of TORC by STS in KC-167 cells was verified by immunoblot assay (data not shown).

(D) CRE-luciferase (CRE-luc) reporter activity in HEK293T cells expressing WT or Ser157Ala mutant Drosophila TORC. Effect of FSK or the dominant-negative CREB inhibitor ACREB on CRE-luc activity is shown. n = 6; p < 0.05. Data are given as means ± SD.

(E and F) Top: immunoblots of Drosophila TORC protein in wild-type (w¹¹¹⁸) flies exposed to water-only starvation (E) or paraquat (PQT) feeding (F). For starvation assay, time after food withdrawal is shown in hours. Flies were maintained on paraquat-containing food for 24 hr before analysis. Phospho- and dephospho-TORC protein is indicated. Bottom: relative effect of starvation or paraquat feeding on TORC protein and mRNA amounts.