A high-resolution systems-wide screen for substrates of the SCFTrCP ubiquitin E3 ligase

<p><em>presented in: HUPO World Congress: The proteome quest to understand biology and disease in Madrid, Spain, 2013</em></p> <p> </p> <p>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, the largest family of enzymes in mammals. Therefore, reliable assays aimed at the identification of substrates of ubiquitin ligases are crucial, not only to unravel the molecular mechanisms by which the UPS controls protein degradation, but also for drug discovery purposes since many established UPS substrates are oncoproteins or tumor suppressors. Here, we develop a combined bioinformatics and affinity purification-mass spectrometry (AP-MS) workflow for identifying in a systems-wide manner bone fide substrates of SCFβTrCP, a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are trademarked by a multi-subunit architecture typically comprising the invariable subunits Rbx1, Cul1, and Skp1 and one of 69 F-box proteins. SCFβTrCP binds, via its WD40 repeats, the DpSGXX(X)pS di-phosphorylated motif in its substrates. Our combined workflow recovers 27 previously reported SCFβTrCP substrates, of which 22 are confidently verified by two independent statistical protocols, confirming the reliability of this approach. Besides known substrates, we identify 221 proteins that, besides harboring the DpSGXX(X)pS motif, also interact specifically with the WD40 repeats. From this list, we highlight several putative novel SCFβTrCP substrates with their putative degron motifs as well as phosphorylation and ubiquitylation sites. Thus, we demonstrate that the integration of structural information, AP-MS and degron motif mining constitutes a generic, specific and effective screen for the identification of substrates of ubiquitin ligases.</p