Background

Regulation of protein degradation is particularly relevant for the cell to control the expression levels of regulatory proteins along with the underlying biological processes. The major known cellular proteostasis mechanism is mediated by the ubiquitin-proteasome system (UPS) and uses a dedicated set of widespread enzymes called ubiquitin E-ligases. Their role is to flag specific proteins by adding a ubiquitin moiety to specific lysine residues that will then be further identified, targeted, and complexed by E-ligases (typically initiated by E3, followed by E2 and E1 types) and ultimately degraded by the UPS (reviewed in [1]). The essential amino-acid sequence allowing ubiquitin-targeted protein degradation has been called Degrons [2–4]. Degrons in which phosphorylation residue(s) play either a permissive or inhibitory role towards degradation are best defined as phospho-(regulated) Degrons and are ubiquitously found in regulatory proteins involved in growth and developmental processes [4,5]. Phospho-Degrons in which the alternative phosphorylation vs. ubiquitylation requirement establishes a biological switch are also referred as phospho-inhibited degrons [6]. Phospho-Degron motifs can be predicted by the parallel presence of in vivo PTM-modified Ser/Thr/Tyr amino-acid residues within a relative short distance from an in vivo modified lysine [7–9]. At present, the post-translational modification (PTM) status of putative Phospho-Degrons can be exerted by independent assays looking either at the ubiquitylation (in vitro ubiquitination assay) or phosphorylation status (through in vitro kinase assays). We have designed an integrated strategy and method to enhance specificity towards both (a) identifying a putative phospho-degron and simultaneously (b) functionally confirm the ID of the ubiquitin E1,2,3-ligases recruited by the phospho-degron-bearing protein/domain. Central to the described method is the synthesis of a biotin-tagged protein domain/subdomain or peptide bearing the native ubiquitin-binding motif amino acidic sequence for the putative phospho-Degron. This recombinant biotin-tagged protein domain/peptide is used as the bait component of the PTM-enhanced (PTMe) pull-down method. The method relies on the ex vivo–enhanced phosphorylation and ubiquitylation (PTMs) events induced in vivo. This bears increased advantages over performing separate co-immunoprecipitations (co-IP)/far westerns and individual kinase or ubiquitylation assays in immunoprecipitated proteins, as shown in Figure 1. The method can indeed be used prior to further refining individual protein interactions within a protein degradation complex, requiring deletion and single-point recombinant mutants, therefore sparing lengthy preparatory work for the expression and purification of the individual recombinant proteins before having clarified their functional involvement in the degradation complex. PTMe pull-down conditions can also be used for proteomics profiling in the degradation complex discovery phase, but the LC-Ms/Ms approach may not be available to all laboratories. We have successfully used this method towards the characterization of a growth factor–regulated phospho-Degron that we initially identified in an angiogenic/metastatic membrane receptor kinase of the Ephrin family, over-expressed in malignant mesothelioma cells [6]. We have adopted the described protocol in combination with immune-enriched proteomics towards further identifying the protein complex for the degradation of the angiogenic kinase EphB4 [6], whose inhibition by an IGF-II autocrine signal determines its over-expression in a malignant mesothelioma cell line [5].

Figure 1. Immuno-blotting detection branch of the post-translational modification–enhanced (PTMe) pull-down method and comparison with traditional co-immune precipitation (co-IP) on the same cellular extract batch from treated vs. untreated cells. An aliquot of the material used in each condition was run on a gel and used for Coomassie stain. The asterisk-marked arrows refer to the complex components for which the method can provide results not observed with traditional co-IP as specified under background. The right-side image is part of the study by Scalia et al. (2023) [6], in which the method was first used. *Please note that two different areas of the same Coomassie gel are shown for comparing the post-normalization protein amount of the same cell extract batches used for both the displayed co-IP and PTMe pull-down experiments.

Figure 2. Membrane partition suggested for parallel analysis of ubiquitin (Ub)-E-ligases detection/pDegron recruitment study using post-translational modification–enhanced (PTMe) pull-down assay. Membrane partition is suggested given the relevance of studying same-cell extract batches deriving from individual in vivo treatment conditions and underlying PTMe pulled-down complexes from individual experiment. Proper control suggested between conditions is a Coomassie stain of equal amounts of (protein-normalized) cell extract input resolved by SDS-PAGE.