Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, 00790, Finland and.
Institute of Biotechnology, University of Helsinki, Helsinki, 00790, Finland.
G3 (Bethesda). 2020 Aug 5;10(8):2843-2849. doi: 10.1534/g3.120.401383.
Insulin/insulin-like growth factor signaling (IIS) is a conserved mechanism to regulate animal physiology in response to nutrition. IIS activity controls gene expression, but only a subset of transcriptional regulators (TRs) targeted by the IIS pathway is currently known. Here we report the results of an unbiased screen for TRs phosphorylated in an IIS-dependent manner. To conduct the screen, we built a library of 857 V5/Strep-tagged TRs under the control of Copper-inducible metallothionein promoter (pMt). The insulin-induced phosphorylation changes were detected by using Phos-tag SDS-PAGE and Western blotting. Eight proteins were found to display increased phosphorylation after acute insulin treatment. In each case, the insulin-induced phosphorylation was abrogated by mTORC1 inhibitor rapamycin. The hits included two components of the NURF complex (NURF38 and NURF55), bHLHZip transcription factor Max, as well as the ortholog of human proliferation-associated 2G4 (dPA2G4). Subsequent experiments revealed that the expression of the gene was promoted by the mTOR pathway, likely through transcription factor Myc. Furthermore, NURF38 was found to be necessary for growth in larvae, consistent with the role of IIS/mTOR pathway in growth control.
胰岛素/胰岛素样生长因子信号（IIS）是一种保守的机制，可调节动物生理学以响应营养。IIS 活性控制基因表达，但目前仅已知 IIS 途径靶向的转录调节剂（TRs）的一部分。在这里，我们报告了一种针对以 IIS 依赖性方式磷酸化的 TRs 的无偏筛选的结果。为了进行筛选，我们构建了一个由 857 个 V5/Strep 标记的 TRs 组成的文库，受铜诱导的金属硫蛋白启动子（pMt）的控制。通过使用 Phos-tag SDS-PAGE 和 Western blot 检测胰岛素诱导的磷酸化变化。发现有 8 种蛋白质在急性胰岛素处理后显示出磷酸化增加。在每种情况下，mTORC1 抑制剂 rapamycin 均可消除胰岛素诱导的磷酸化。命中物包括 NURF 复合物的两个成分（NURF38 和 NURF55）、bHLHZip 转录因子 Max 以及人类增殖相关 2G4（dPA2G4）的同源物。随后的实验表明，基因的表达受 mTOR 途径促进，可能通过转录因子 Myc。此外，发现 NURF38 对于幼虫的生长是必需的，这与 IIS/mTOR 途径在生长控制中的作用一致。
