Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin
Figure 7
Activation and enzymatic function of Parkin.
A) HeLa cells were transfected with FLAG-Parkin C431S. Cells were treated with CCCP for 0, 1, 2, 4, or 16 h and harvested. Western blots were prepared to monitor the formation of a Parkin C431S-Ub oxyester, which, in contrast to unmodifed Parkin (closed arrowhead) appears as a band shift (open arrowhead) and is sensitive to NaOH treatment. The phospho-mimic mutations S65D or S65E showed some levels of Parkin C431S-Ub even in the absence of CCCP, consistent with a slight activation under steady-state conditions. B) HEK293E cells were transfected with FLAG-Parkin wild type and left either untreated or were treated with 10 µM CCCP. FLAG immunoprecipitations were performed and ubiquitinations reactions were carried out on the beads. All ubiquitination reactions contained E1, ATP, and N-terminally biotinylated Ub. Either no E2 enzyme, or UBE2D2, UBE2L3 or UBE2N plus its co-factor Uev1a were added. In order to analyze the effect of Parkin phosphorylation, some FLAG immunoprecipitates were pretreated with phosphatase before Ub reaction was carried out. UBE2D2, UBE2L3 and UBE2N can serve as co-factors for Parkin in vitro. CCCP treatment is not required, but enhances the ubiquitination reactions. Phosphatase treatment reduces the ubiquitinations to the extent observed without CCCP treatment. UBE2N shows no detectable activity towards Parkin auto-ubiquitination. A closed arrowhead indicates the position of unmodified FLAG-Parkin, an open arrowhead labels Ub modified species. An arrow labels the molecular weight of Parkin on the Streptavidin-HRP blot.