Direct Monitoring of the Inhibition of Protein–Protein Interactions in Cells by Translocation of PKCδ Fusion Proteins

Abstract

Seeing is believing: When a bait protein is fused to protein kinase C (PKCδ), the proteins can interact and cotranslocate from the cytoplasm to the plasma membrane (see picture; imaging with red and green fluorescent proteins (RFP and GFP)). In contrast, when the bait–target interaction is inhibited, only the bait protein is translocated to the plasma membrane. This method was applied to several protein pairs.

A small-molecule inhibitor targeting the AURKC-IκBα interaction decreases transformed growth of MDA-MB-231 breast cancer cells

“…To identify the binding partner of AURKC, we performed a cellular protein translocation–based screen using a GFP-tagging vector library in HEK293T cells. The library consists of GFP fusions of 597 kinases and kinase-related genes, and was designed for analysis of PPI in cells by laser scanning fluorescence microscopy [ 18 , 19 ]. We focused the screen on kinases because they represent a set of readily druggable targets related to AURKC that are potentially amenable to clinical translation.…”

“…Cells were treated with AKCI and then trypsinized, washed with PBS, and subjected to cell-cycle analysis on a NucleoCounter NC-3000 (ChemoMetec, Allerød, Denmark). Two-step analysis was performed in which the cells were lysed and the nuclei were stained with DAPI [ 18 ].…”

PCNA Unloading Is Negatively Regulated by BET Proteins

“…To examine the intracellular interaction between ATAD5 and BRD4, we used cell-based unidentified protein interaction discovery (CUPID) analysis (Lee et al, 2011). The CUPID analysis has been used for real-time monitoring of protein-protein interactions.…”

Rapidly Reversible Manipulation of Molecular Activity with Dual Chemical Dimerizers

“…[8] In one study, FK506 reversed rapamycin-induced dimerization reasonably well on a timescale of 10 min in HEK-293T cells. [9] To test the efficiency of this competition strategy in our dimerization system, we first treated cells with rapamycin to induce the rapid relocation of CFP-FRB from cytosol to the Lyn-YFP-FKBP-expressing plasma membrane. Subsequently, we added increasing concentrations of FK506 to compete with the interaction between FKBP and rapamycin.…”