Abstract
BackgroundGenetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways.ResultsOur biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia.ConclusionsOur study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways converge at GSK3β to control Myc protein stability, while our genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia.
Highlights
Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth
Insulin induces Myc protein accumulation in Drosophila S2 cells, with a mechanism that results in inactivation of glycogen synthase kinase 3beta (GSK3b) and is dependent on target of rapamycin (TOR) complex 1 activity Our previous observation that Myc protein stability in Drosophila S2 cells is reduced by GSK3b activity [10] led us to investigate if stimulation of insulin signaling, which inhibits GSK3b via Akt phosphorylation, could result in Myc protein accumulation
Myc protein accumulation by insulin was accompanied by phosphorylation of Akt on Ser 505, an event that correlated with phosphorylation of GSK3b on Ser 9 (Figure 1B), and was inhibited in the presence of the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (Figure 1C)
Summary
Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Genetic studies in Drosophila have identified Myc as well as components of insulin and target of rapamycin (TOR) signaling pathways as key regulators of growth [1,2]. Insulin and TOR activities are balanced by a negative feedback mechanism that is activated when S6K is hyperactivated to counteract insulin activity Under this condition, S6K phosphorylates IRS1-4/chico triggering its internalization and subsequent proteasomal degradation [16,17]. This feedback mechanism is reduced in pathological conditions, such as TSC syndromes where cells carrying mutations in tsc or tsc display an abnormal increase in size and exhibit constitutive phosphorylation of S6K [18] In these cells, hyper-activation of S6K correlates with inactivation of GSK3b by phosphorylation of Serine 9, which results in c-Myc protein accumulation [18]
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