Insulin-induced protein phosphorylation in imaginal wing discs of Drosophila melanogaster

Insulin or insulin-like signal has been well-known for their roles in regulating glucose uptake, cell size, organ size, and cell proliferation in many multicellular organisms. In order to study its working mechanism in Drosophila, insulin-induced protein phosphorylation in the imaginal wing disc cells was monitored by measuring radioactive orthophosphate incorporation to the endogenous proteins. Insulin stimulated the phosphorylation of 34 kDa protein, a putative ribosomal protein S6, in the imaginal wing discs. Phosphorylation of the 34 kDa protein was observed as early as 15 min after insulin treatment and the increment of phosphorylation level was insulin dose-dependent. In vitro S6 kinase assay showed that the kinase activity in the imaginal wing discs was dramatically increased by insulin stimulation. Specific kinase inhibitors, rapamycin for TOR (target of rapamycin) and wortmannin for PI3K (phosphoinositide 3-kinase), strongly inhibited the insulin-stimulated phosphorylation of 34 kDa protein and S6 kinase activity. In contrast, okadaic acid, a phosphatase 1 and 2A inhibitor, was able to partially overcome the inhibition of 34 kDa protein phosphorylation by rapamycin and wortmannin. These data suggest the conservation of the insulin-dependent S6 kinase signaling pathway in the imaginal wing discs of Drosophila.