A systems-wide screen identifies substrates of the SCF ^βTrCP 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.

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

“…A similar approach combined bioinformatics and AP–MS/MS to identify SCF βTrCP E3 ligase substrates. First, a consensus motif derived from reported βTrCP substrates was generated and used to search biological sequence databases for substrates [92]. Subsequently, using AP–MS/MS, βTrCP-interacting proteins were identified and the data were integrated with the bioinformatics prediction data [92].…”

“…First, a consensus motif derived from reported βTrCP substrates was generated and used to search biological sequence databases for substrates [92]. Subsequently, using AP–MS/MS, βTrCP-interacting proteins were identified and the data were integrated with the bioinformatics prediction data [92]. This approach identified 221 βTrCP interactors that also harbour conserved sequences similar to the consensus motif of known substrates [92].…”

“…Subsequently, using AP–MS/MS, βTrCP-interacting proteins were identified and the data were integrated with the bioinformatics prediction data [92]. This approach identified 221 βTrCP interactors that also harbour conserved sequences similar to the consensus motif of known substrates [92]. …”

E3 Ligase SCFβTrCP-induced DYRK1A Protein Degradation Is Essential for Cell Cycle Progression in HEK293 Cells

“…SCF βTrCP was the most frequently studied ubiquitin ligase. It could specifically bind to the DpSG XX pS motif, where serine residues were phosphorylated, and subsequently mediated by ubiquitination of substrates (28, 29). Ubiquitin cluster covalently connected to substrates functioned as a degradation signal and could be recognized by proteasome for degradation (26).…”