Kim Tai Young, Siesser Priscila F, Rossman Kent L, Goldfarb Dennis, Mackinnon Kathryn, Yan Feng, Yi XianHua, MacCoss Michael J, Moon Randall T, Der Channing J, Major Michael B
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Mol Cell Biol. 2015 Jan;35(1):167-81. doi: 10.1128/MCB.00857-14. Epub 2014 Oct 20.
Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2/FBXW11, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to "trap" ubiquitylated substrates on the SCF(FBXW11) E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCF(FBXW11) bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.
确定每个泛素连接酶的完整底物库仍然是一项重大挑战。质谱仪器、计算方法以及蛋白质生物化学方法的改进催生了几种用于鉴定泛素连接酶底物的新方法。在此，我们采用平行适配体捕获（PAC）蛋白质组学方法来研究βTrCP2/FBXW11，它是SKP1-CUL1-F盒（SCF）E3泛素连接酶复合物的底物适配体。泛素化反应的持续性要求FBXW11与其底物之间进行短暂的物理相互作用，因此对与FBXW11结合的底物进行生化纯化颇具难度。利用基于PAC的方法，我们抑制蛋白酶体，以便在SCF(FBXW11) E3复合物上“捕获”泛素化底物。对蛋白酶体抑制前后免疫纯化的FBXW11蛋白复合物进行比较质谱分析，发现了21种已知底物和23种可能的新底物。在重点研究中，我们发现SCF(FBXW11)结合、多聚泛素化并使RAPGEF2不稳定，RAPGEF2是一种激活小GTP酶RAP1的鸟嘌呤核苷酸交换因子。高RAPGEF2蛋白水平促进细胞-细胞融合，进而导致多核化。令人惊讶的是，这一过程独立于鸟嘌呤核苷酸交换因子（GEF）的催化活性以及RAP1的存在。我们的数据确立了RAPGEF2的新功能，这些功能可能与癌症中的非整倍体形成有关。更广泛地说，本报告支持继续使用底物捕获蛋白质组学来全面定义E3泛素连接酶的靶标。所有蛋白质组学数据可通过ProteomeXchange获取，标识符为PXD001062。
