title: A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase.
abstract: Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases. Identification of substrates of ubiquitin ligases is crucial not only to unravel the molecular mechanisms by which the UPS controls protein degradation but also for drug discovery purposes because many established UPS substrates are implicated in disease. We developed a combined bioinformatics and affinity purification-mass spectrometry (AP-MS) workflow for the system-wide identification of substrates of SCF(βTrCP), a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are characterized by a multisubunit architecture typically consisting of the invariable subunits Rbx1, Cul1, and Skp1 and one of 69 F-box proteins. The F-box protein of this member of the family is βTrCP. SCF(βTrCP) binds, through the WD40 repeats of βTrCP, to the DpSGXX(X)pS diphosphorylated motif in its substrates. We recovered 27 previously reported SCF(βTrCP) substrates, of which 22 were verified by two independent statistical protocols, thereby confirming the reliability of this approach. In addition to known substrates, we identified 221 proteins that contained the DpSGXX(X)pS motif and also interacted specifically with the WD40 repeats of βTrCP. Thus, with SCF(βTrCP), as the example, we showed that integration of structural information, AP-MS, and degron motif mining constitutes an effective method to screen for substrates of ubiquitin ligases.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands. Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, Netherlands.
authors.forename: Teck Yew
authors.initials: TY
authors.lastname: Low
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands. Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, Netherlands.
authors.forename: Mao
authors.initials: M
authors.lastname: Peng
authors.affiliation: Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, Netherlands.
authors.forename: Roberto
authors.initials: R
authors.lastname: Magliozzi
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands. Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, Netherlands.
authors.forename: Shabaz
authors.initials: S
authors.lastname: Mohammed
authors.affiliation: Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, Netherlands.
authors.forename: Daniele
authors.initials: D
authors.lastname: Guardavaccaro
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands. Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, Netherlands. a.j.r.heck@uu.nl.
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D020816:Amino Acid Motifs; D057809:HEK293 Cells; D006801:Humans; D044843:SKP Cullin F-Box Protein Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D044844:beta-Transducin Repeat-Containing Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D044844:beta-Transducin Repeat-Containing Proteins; D044843:SKP Cullin F-Box Protein Ligases