Insulin regulates an essential conserved signaling pathway affecting growth, proliferation, and metabolism. To expand our understanding of the insulin pathway, we combine biochemical, genetic, and computational approaches to build a comprehensive Drosophila InR/PI3K/Akt network. First, we map the dynamic protein-protein interaction network surrounding the insulin core pathway using bait-prey interactions connecting 566 proteins. Combining RNAi screening and phospho-specific antibodies, we find that 47% of interacting proteins affect pathway activity, and, using quantitative phosphoproteomics, we demonstrate that 10% of interacting proteins are regulated by insulin stimulation at the level of phosphorylation. Next, we integrate these orthogonal datasets to characterize the structure and dynamics of the insulin network at the level of protein complexes and validate our method by identifying regulatory roles for the Protein Phosphatase 2A (PP2A) and Reptin-Pontin chromatin-remodeling complexes as negative and positive regulators of ribosome biogenesis, respectively. Altogether, our study represents a comprehensive resource for the study of the evolutionary conserved insulin network.
Drosophila larval skeletal muscles are single, multinucleated cells of different sizes that undergo tremendous growth within a few days. The mechanisms underlying this growth in concert with overall body growth are unknown. We find that the size of individual muscles correlates with the number of nuclei per muscle cell and with increasing nuclear ploidy during development. Inhibition of Insulin receptor (InR; Insulin-like receptor) signaling in muscles autonomously reduces muscle size and systemically affects the size of other tissues, organs and indeed the entire body, most likely by regulating feeding behavior. In muscles, InR/Tor signaling, Foxo and dMyc (Diminutive) are key regulators of endoreplication, which is necessary but not sufficient to induce growth. Mechanistically, InR/Foxo signaling controls cell cycle progression by modulating dmyc expression and dMyc transcriptional activity. Thus, maximal dMyc transcriptional activity depends on InR to control muscle mass, which in turn induces a systemic behavioral response to allocate body size and proportions.