Vinayagam Arunachalam, Kulkarni Meghana M, Sopko Richelle, Sun Xiaoyun, Hu Yanhui, Nand Ankita, Villalta Christians, Moghimi Ahmadali, Yang Xuemei, Mohr Stephanie E, Hong Pengyu, Asara John M, Perrimon Norbert
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Department of Computer Science, Volen Center for Complex Systems, Brandeis University, Waltham, MA 02454, USA.
Cell Rep. 2016 Sep 13;16(11):3062-3074. doi: 10.1016/j.celrep.2016.08.029.
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.
胰岛素调节着一条影响生长、增殖和代谢的重要保守信号通路。为了拓展我们对胰岛素通路的理解，我们结合生化、遗传和计算方法构建了一个全面的果蝇胰岛素受体（InR）/磷脂酰肌醇-3激酶（PI3K）/蛋白激酶B（Akt）网络。首先，我们利用连接566种蛋白质的诱饵-猎物相互作用，绘制了围绕胰岛素核心通路的动态蛋白质-蛋白质相互作用网络。结合RNA干扰筛选和磷酸化特异性抗体，我们发现47%的相互作用蛋白影响通路活性，并且通过定量磷酸化蛋白质组学，我们证明约10%的相互作用蛋白在磷酸化水平受胰岛素刺激调控。接下来，我们整合这些正交数据集，在蛋白质复合物水平表征胰岛素网络的结构和动态，并通过分别鉴定蛋白磷酸酶2A（PP2A）和Reptin-Pontin染色质重塑复合物作为核糖体生物发生的负调控因子和正调控因子的调节作用来验证我们的方法。总之，我们的研究为进化保守的胰岛素网络研究提供了一个全面的资源。
