Figure 1.

Insulin induces Myc protein upregulation in Drosophila S2 cells, which depends on TOR signaling. (A) Time course of Myc protein accumulation upon insulin treatment. Insulin was added to serum-starved S2 cells and Myc levels were analyzed by western blotting. (B) Insulin treatment increases Myc protein levels, correlating with phosphorylation of Akt on Ser 505 and of GSK3β on Ser 9. (C) Myc protein accumulation by insulin is inhibited in the presence of the PI3K inhibitor wortmannin. (D and E). Blocking GSK3β activity with LiCl or using the GSK3β kinase dead mutant (GSK3β-KD), affects insulin-induced Myc protein accumulation. To block GSK3β activity, S2 cells were treated with LiCl (D), or transfected with a plasmid encoding for an HA-tagged GSK3β-KD that was previously demonstrated to reduce endogenous GSK3β kinase activity [10] (E). Treatment with insulin results in the accumulation of Myc protein (D and E, lane 2). Blocking GSK3β activity by LiCl (panel D, lane 3) or by the expression of GSK3β-KD (panel E, lane 3) enhanced Myc protein level, which was not further increased when insulin was added to cells where GSK3β activity was inhibited by LiCl or by the presence of its KD mutant (lane 4). Expression of the HA-GSK3β-KD was analyzed by western blotting using anti-HA antibodies. (F) Inhibition of TORC1 by rapamycin decreased insulin-induced Myc protein accumulation. Rapamycin, alone or together with insulin, was added for 2 h to serum-starved S2 cells; vinculin was used as a control for protein loading. (G) Blocking the proteosome pathway with MG132 inhibits Myc protein degradation by rapamycin. MG132 was added to the cells together with rapamycin. Myc protein levels were analyzed after 2 hours of treatment using anti-Myc antibodies; actin was used as a control for protein loading. GSK3β: glycogen synthase kinase 3-beta; LiCl: lithium chloride; KD: kinase dead; PI3K: phosphatidyl-inositol-3 kinase; TORC1: target of rapamycin complex 1.

Parisi et al. BMC Biology 2011 9:65   doi:10.1186/1741-7007-9-65
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