An Integrative Analysis of the InR/PI3K/Akt Network Identifies the Dynamic Response to Insulin Signaling

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. [Display omitted] •The dynamic protein-protein interaction network around the insulin pathway is mapped•Half of the network components are validated using RNAi screens•Approximately 10% of interacting proteins are phospho-regulated by insulin stimulation•PP2A and Reptin-Pontin modulate the insulin pathway Vinayagam et al. combine biochemical, genetic, and computational approaches to build a comprehensive Drosophila insulin signaling network that connects more than 500 proteins to the canonical pathway. Characterizing the structure and dynamics of this network revealed 143 dynamic protein complexes that are essential for insulin signaling.