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Substrate trapping proteomics reveals targets of the ?TrCP2/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.

SUBMITTER: Kim TY

PROVIDER: S-EPMC4295375 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Substrate trapping proteomics reveals targets of the βTrCP2/FBXW11 ubiquitin ligase.

Kim Tai Young TY Siesser Priscila F PF Rossman Kent L KL Goldfarb Dennis D Mackinnon Kathryn K Yan Feng F Yi XianHua X MacCoss Michael J MJ Moon Randall T RT Der Channing J CJ Major Michael B MB

Molecular and cellular biology 20141020 1

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 rea ...[more]

PMID: 25332235

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Project description:BackgroundTumor metastasis is a major factor for poor prognosis of hepatocellular carcinoma (HCC), but the relationship between ubiquitination and metastasis need to be studied more systematically. We analyzed the ubiquitinome of HCC in this study to have a more comprehensive insight into human HCC metastasis.MethodsThe protein ubiquitination levels in 15 HCC specimens with vascular invasion and 15 without vascular invasion were detected by ubiquitinome. Proteins with significantly different ubiquitination levels between HCCs with and without vascular invasion were used to predict E3 ubiquitin ligases associated with tumor metastasis. The topological network of protein substrates and corresponding E3 ubiquitin ligases was constructed to identify the key E3 ubiquitin ligase. Besides, the growth, migration and invasion ability of LM3 and HUH7 hepatoma cell lines with and without SYVN1 expression interference were measured by cell proliferation assay, subcutaneous tumor assay, umphal vein endothelium tube formation assay, transwell migration and invasion assays. Finally, the interacting proteins of SYVN1 were screened and verified by protein interaction omics, immunofluorescence, and immunoprecipitation. Ubiquitin levels of related protein substrates in LM3 and HUH7 cells were compared in negative control, SYVN1 knockdown, and SYVN1 overexpression groups.ResultsIn this study, our whole-cell proteomic dataset and ubiquitinomic dataset contained approximately 5600 proteins and 12,000 ubiquitinated sites. We discovered increased ubiquitinated sites with shorter ubiquitin chains during the progression of HCC metastasis. In addition, proteomic and ubiquitinomic analyses revealed that high expression of E3 ubiquitin-protein ligase SYVN1 is related with tumor metastasis. Furthermore, we found that SYVN1 interacted with heat shock protein 90 (HSP90) and impacted the ubiquitination of eukaryotic elongation factor 2 kinase (EEF2K).ConclusionsThe ubiquitination profiles of HCC with and without vascular invasion were significantly different. SYVN1 was the most important E3 ubiquitin-protein ligase responsible for this phenomenon, and it was related with tumor metastasis and growth. Therefore, SYVN1 might be a potential therapeutic target for HCC.

Dataset Information

Substrate trapping proteomics reveals targets of the ?TrCP2/FBXW11 ubiquitin ligase.

Publications

Substrate trapping proteomics reveals targets of the βTrCP2/FBXW11 ubiquitin ligase.

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