Unique auto-ubiquitination activities of artificial RING fingers in cancer cells.

Ubiquitin-conjugating (E2) enzymes in protein ubiquitination are associated with various diseases. An artificial RING finger (ARF) is a useful tool, and E2 activities are conveniently estimated based on ARF reactivities. To extend the use of ARF in cells, we constructed a TAT-ARF using a cell-penetrating trans-activator protein (TAT) peptide. An in vitro ubiquitination assay without substrates showed auto-ubiquitination of TAT-ARF via its TAT region. TAT-ARF was translocated into MCF7 breast cancer cells, and then TAT-ARF ubiquitinated itself via its ARF. Experiments using confocal laser-scanning microscopy revealed that FAM-labeled TAT-ARF was readily internalized in cells and it remained encapsulated in vesicles. The Cell Counting Kit-8 assay indicated that the TAT-ARF uptake occurred without cytotoxicity in MCF7 cells at concentrations below 5.0 μM. By taking advantage of TAT-ARF, we, for the first time, succeeded in detecting E2 activities in cells. Thus, the present work opens up new avenues in the investigation of protein ubiquitination.