Abstract
The identification and characterization of the ubiquitin E-ligase complexes involved in specific proteins' degradation via the ubiquitin-proteasome system (UPS) can be challenging and require biochemical purification processes and in vitro reconstitution assays. Likewise, evaluating the effect of parallel phosphorylation and ubiquitination events occurring in vivo at dual phospho/ubiquitin-regulated motifs (called Phospho-Degrons or pDegrons) driving UPS degradation of the targeted protein has remained elusive. Indeed, the functional study of such E1-E2-E3 complexes acting on a protein-specific level requires previously or otherwise acquired knowledge of the nature of such degradation complex components. Furthermore, the molecular basis of the interaction between an E3 ligase and its pDegron binding motif on a target protein would require individually optimized in vitro kinase and ubiquitination assays. Here, we describe a novel enzymatically enhanced pull-down method to functionally streamline the discovery and functional validation of the ubiquitin E-ligase components interacting with a phospho-degron containing protein domain and/or sub-domain. The protocol combines key features of a protein kinase and ubiquitination in vitro assay by including them in a pull-down step exerted by a known or putative pDegron-tagged peptide using the cell extracts as a source of enzymatically active post-translational modification (PTM) modifying/binding native proteins. The same method allows studying specific stimuli or treatments towards the recruitment of the molecular degradation complex at the target protein's phospho-degron site, reflecting in vivo-initiated events further enhanced through the assay design. In order to take full advantage of the method over traditional protein-protein interaction methods, we propose to use this PTM-enhanced (PTMe) pull down both towards the degradation complex discovery/ID phase as well as for the functional pDegron recruitment validation phase, which is the one described in the present protocol both graphically and in a stepwise fashion for reproduceable results. Key features • Suitable to study UPS-regulated (a) cytosolic and/or nuclear proteins, (b) intracellular region of transmembrane proteins, and (c) protein sub-domains bearing a known/putative pDegron motif. • Requires a biotin-tagged recombinant version of the target protein and/or sub-domain. • Allows the qualitative and quantitative analysis of endogenous ubiquitin (Ub) E-ligases recruitment to a known or putative pDegron bearing protein/sub-domain. • Allows simultaneous testing of various treatments and/or conditions affecting the phosphorylative and/or ubiquitylation status of the studied pDegron bearing protein/sub-domain and the recruited factors. Graphical overview.