Coyaud Etienne, Mis Monika, Laurent Estelle M N, Dunham Wade H, Couzens Amber L, Robitaille Melanie, Gingras Anne-Claude, Angers Stephane, Raught Brian
From the ‡Princess Margaret Cancer Centre, University Health Network;
§Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto;
Mol Cell Proteomics. 2015 Jul;14(7):1781-95. doi: 10.1074/mcp.M114.045658. Epub 2015 Apr 21.
The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCF(β-TrCP1) and SCF(β-TrCP2) are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFκB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCF(β-TrCP1/2) interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCF(β-TrCP1/2) substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for β-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that β-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases.
泛素E3连接酶底物的鉴定一直具有挑战性，部分原因在于其低亲和力、瞬时相互作用、靶标的快速降解以及无法从难溶性细胞区室中鉴定蛋白质。SCF(β-TrCP1)和SCF(β-TrCP2)是经过充分研究的泛素E3连接酶，它们将底物靶向蛋白酶体降解，并在Wnt、Hippo和NFκB信号传导中发挥重要作用。通过将26S蛋白酶体抑制剂（MG132）处理与邻近依赖性生物素标记（BioID）和半定量质谱相结合，我们在此鉴定了SCF(β-TrCP1/2)相互作用伴侣。基于它们在MG132存在下的富集情况，我们的数据鉴定出50多个新的假定SCF(β-TrCP1/2)底物。我们验证了其中12种新底物，并揭示了β-TrCP在维持核膜完整性、加工（P）小体周转和翻译控制方面以前未被怀疑的作用。总之，我们的数据表明β-TrCP是细胞应激反应中的一个重要枢纽。本文介绍的技术是对更标准的免疫沉淀-质谱（IP-MS）方法的一种补充方法，应该广泛适用于鉴定许多泛素E3连接酶的底物。
