SCF-Skp2 E3 Ligase: p27 Protein Degradation Redistribution Assay

• Designed to assay compounds for their ability to modulate activation of SCF-Skp2 ubiquitin E3 ligase activity
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Conjugation of ubiquitin (a 76 aa protein) to a protein substrate during protein degradation by the proteasome follows a three-step mechanism. First, the ubiquitin activating enzyme E1 activates ubiquitin to generate a high-energy thiol-ester intermediate in a reaction dependent on ATP. The activated ubiquitin is then transferred to a member of the E2 ubiquitin conjugating enzymes, that finally transfers the ubiquitin to a substrate protein that is specifically bound by an E3 ligase enzyme. One such E3 ligase is the SCF-Skp2 complex that belongs to the RING finger class of E3 ubiquitin ligases. SCF consists of three primary subunits (Skp1, Cullin and Rbx/Roc1) that are responsible for binding to E2 enzyme and to so-called F-box proteins that recruit protein substrates and thereby bridge the reaction between the E2 ubiquitin conjugating enzyme and the substrate. The SCF-Skp2 E3 ligase is mainly involved in promoting cell cycle progression, most notably through targeted degradation of p27Kip1 phosphorylated at Thr187 in S-phase. Furthermore, Skp2 has been found to promote and be upregulated in a number of human cancers.

Figure 1. Inhibition of proteasomal degradation of p27Kip1(T187D)-EGFP. Cells were transfected with siRNAs or treated with the proteasome inhibitor MG132. Knockdown of Skp2 E3 ligase or treatment with MG-132 results in stabilization of p27 (T187D)-EGFP, while knockdown of other E3 ligases results in no (βTRCP) or low (E6-AP) accumulation. EGFP siRNA is the positive control for efficiency of siRNA transfection.

Figure 2. MG-132 concentration response in the SCF-Skp2 E3 Ligase:p27 Protein Degradation assay. The EC₅₀ is ~3 μM. Concentration response was measured in 9 point half log dilution series (n=8). Cells were treated with test compound for 24 hr. Cells were then fixed and increase in fluorescence intensity was measured using the Cellomics ArrayScan V^TI Reader and the Molecular Translocation V2 BioApplication. % activity was calculated relative to the positive (5 μM MG-132) and negative control (0.25% DMSO).