title: 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 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.
authors.affiliation: 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.
authors.forename: Tai Young
authors.initials: TY
authors.lastname: Kim
authors.affiliation: 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.
authors.forename: Priscila F
authors.initials: PF
authors.lastname: Siesser
authors.affiliation: 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.
authors.forename: Kent L
authors.initials: KL
authors.lastname: Rossman
authors.affiliation: Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Dennis
authors.initials: D
authors.lastname: Goldfarb
authors.affiliation: 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.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Mackinnon
authors.affiliation: 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.
authors.forename: Feng
authors.initials: F
authors.lastname: Yan
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: XianHua
authors.initials: X
authors.lastname: Yi
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
authors.affiliation: Department of Pharmacology and HHMI, University of Washington, Seattle, Washington, USA.
authors.forename: Randall T
authors.initials: RT
authors.lastname: Moon
authors.affiliation: 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.
authors.forename: Channing J
authors.initials: CJ
authors.lastname: Der
authors.affiliation: 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 Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA ben_major@med.unc.edu.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Major
mesh_terms: D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D020543:Proteome; D040901:Proteomics; D034741:RNA, Small Interfering / Q000378:metabolism; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D044844:beta-Transducin Repeat-Containing Proteins / Q000502:physiology*
chemical_list: C471645:FBXW11 protein, human; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; D020543:Proteome; C403525:RAPGEF2 protein, human; D034741:RNA, Small Interfering; D000089804:Shelterin Complex; C440809:TERF2IP protein, human; D034501:Telomere-Binding Proteins; D025801:Ubiquitin; D044844:beta-Transducin Repeat-Containing Proteins; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex