Substrate Trapping Proteomics Reveals Targets of the βTrCP2/FBXW11 Ubiquitin Ligase

Abstract

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 ␤TrCP2/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. U biquitylation is a posttranslational modification that controls protein-protein interactions, protein subcellular localization, protein-mediated catalysis, and, most famously, protein stability. The enzymology of protein ubiquitylation is now fairly well understood and has been well summarized in several recent reviews (1-3). The last and arguably most important step in the ubiquitylation reaction is carried out by an E3 ubiquitin ligase. These proteins select substrates for ubiquitylation, physically bridge and orient the substrate with ubiquitin, and in some cases, directly catalyze ubiquitin transfer. E3 ligases also provide the cell with a means to dynamically regulate substrate ubiquitylation; the interaction of a substrate protein with its cognate E3 ligase is often influenced by peripheral signals, such as phosphorylation (4). In total, more than 600 distinct E3 ubiquitin ligases have been identified within the human genome (5), the vast majority of which remain unstudied. Current estimates suggest that these ligases target more than 9,000 distinct human proteins for ubiquitylation, or roughly 40% of the protein-coding human genome (6, 7). For most of these proteins, the physiological importance of ubiquitin conjugation is not known. Likewise, paired relationships between specific E3 ligases and substrates are for the most part not known.Until recently, substra...

The nonreceptor tyrosine kinase c-Src attenuates SCF(β-TrCP) E3-ligase activity abrogating Taz proteasomal degradation

“…RNAi targeting TrCP1/2 was from Sigma, directed against the sequence GUGGAAUUUGUGGAACAUC, previously reported (32). siGENOME Control siRNA (nontargeting siRNA #5) was used as nontargeting control.…”

BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates*

“…Substrates-While our work was in progress, two additional studies were published on the identification of ␤-TrCP substrates (56,57), utilizing more standard IP-MS and TAP (tandem affinity purification)-MS approaches. Notably, despite the use of widely disparate approaches, many of the same ␤-TrCP interactors were detected among all three studies, significantly expanding the number of high-confidence ␤-TrCP substrates and implicating this already well-studied E3 ligase in several new biological processes.…”

Systematic approaches to identify E3 ligase substrates

“…Furthermore, CompPASS can identify proteins based on a single peptide, increasing the potential for incorrect protein identifications or false-positives [89]. Importantly, PAC-based approaches are unlikely to enrich for monoubiquitylated substrates and substrates that are not fated to be degraded [90]. The PAC–CompPASS approach was recently applied to identify substrates of the FBXW11 ubiquitin ligase adaptor protein [90].…”

“…Importantly, PAC-based approaches are unlikely to enrich for monoubiquitylated substrates and substrates that are not fated to be degraded [90]. The PAC–CompPASS approach was recently applied to identify substrates of the FBXW11 ubiquitin ligase adaptor protein [90]. This study identified 96 interacting proteins, 23 of which are putative substrates, with 1 independently validated as a substrate [90].…”