title: BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates.
abstract: 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.
authors.affiliation: From the ‡Princess Margaret Cancer Centre, University Health Network;
authors.forename: Etienne
authors.initials: E
authors.lastname: Coyaud
authors.affiliation: §Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto;
authors.forename: Monika
authors.initials: M
authors.lastname: Mis
authors.affiliation: From the ‡Princess Margaret Cancer Centre, University Health Network;
authors.forename: Estelle M N
authors.initials: EM
authors.lastname: Laurent
authors.affiliation: ¶Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital;
authors.forename: Wade H
authors.initials: WH
authors.lastname: Dunham
authors.affiliation: ¶Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital;
authors.forename: Amber L
authors.initials: AL
authors.lastname: Couzens
authors.affiliation: §Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto;
authors.forename: Melanie
authors.initials: M
authors.lastname: Robitaille
authors.affiliation: ¶Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital; ‖Department of Molecular Genetics, University of Toronto;
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
authors.affiliation: §Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto; **Department of Biochemistry, University of Toronto;
authors.forename: Stephane
authors.initials: S
authors.lastname: Angers
authors.affiliation: From the ‡Princess Margaret Cancer Centre, University Health Network; ‡‡Department of Medical Biophysics, University of Toronto, Toronto, ON Canada brian.raught@uhnres.utoronto.ca.
authors.forename: Brian
authors.initials: B
authors.lastname: Raught
mesh_terms: D001710:Biotin / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism; D009685:Nuclear Envelope / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D055550:Protein Stability; D015203:Reproducibility of Results; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins / Q000378:metabolism*
chemical_list: D015852:Eukaryotic Initiation Factor-2; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C413025:SUN2 protein, human; D025801:Ubiquitin; D044844:beta-Transducin Repeat-Containing Proteins; D001710:Biotin; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D054645:Protein Phosphatase 1