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. 2009 Oct 29;28(24):3879–3892. doi: 10.1038/emboj.2009.312

Figure 5.

Figure 5

TORC2 is essential for the maximal activation of Trc. (A) Schematic representation of the Trc domain structure and phosphorylation sites. HM indicates the hydrophobic motif, which is highly conserved in the NDR kinase family. (B) Trc phosphorylation of Thr449 but not Ser292 was reduced in sin1, rictor, and Tor mutant embryos. Embryos (stage 16/17) homozygous for sin1PBac, rictorΔ2, TorΔP, S6Kl−1, and Akt1 were selected by their lack of a GFP-expressing balancer chromosome, and then homogenized in sample buffer, boiled, and analyzed by immunoblotting with the indicated antibodies. (C) Knockdown of Sin1, Rictor, or Tor inhibits Trc activation in Drosophila S2 cells. Different dsRNAs used in the experiments are indicated. ‘Control' denotes control dsRNA targeting EGFP. S2 cells were treated with 100 nM okadaic acid (OA+) or with solvent alone (OA−) for 30 min before harvesting where indicated. Cell lysates were analyzed by immunoblotting for the phosphorylation level of Akt (Ser505 for TORC2 activity) and Trc (Thr449). The bottom panel indicates Trc kinase activity relative to the control (‘control ‘ without OA treatment). The kinase activity was determined using the NDR kinase substrate peptides. Error bars indicate the mean±s.d. (n=3). (D) Prolonged treatment of cells with rapamycin inhibits Trc activation. S2 cells were treated with 100 nM rapamycin for the indicated times. Cell lysates were then analyzed by immunoblotting for phosphorylated S6K (Thr398 for TORC1 activity), Akt (Ser505 for TORC2 activity), and Trc (Thr449). The bottom panel indicates Trc kinase activity relative to the control (0 h treatment). (E) TORC2 and Hpo regulate Trc activity through different pathways. S2 cells were incubated with dsRNA against the indicated genes for 7 days and then treated with 100 nM okadaic acid (OA+) or with solvent alone (OA−) for 30 min before harvesting. Cell lysates were then analyzed by immunoblotting for the phosphorylated Akt (Ser505 for TORC2 activity), Hpo (Thr195 for Hpo activity), and Trc (Thr449). Bottom panel indicates Trc kinase activity relative to the control (‘control' without OA treatment). Error bars indicate the mean±s.d. (n=3).