PINK1-dependent phosphorylation of ubiquitin at Ser65 leads to increased activity of ΔUbl-Parkin
(A) MBP–TcPINK1 enhances ΔUbl-Parkin-mediated ubiquitin discharge from UbcH7. ΔUbl-Parkin was phosphorylated using wild-type (WT), kinase-inactive (KI) or no MBP–TcPINK1 in the presence of Mg2+-[γ-32P]ATP. An E2-discharge assay was established by incubation of this mixture with 2 μg of UbcH7 that had been pre-incubated with E1 and FLAG–ubiquitin in the presence of ATP for 60 min. Reactions were allowed to continue for 15 min and were stopped using SDS loading buffer in the absence of reducing agent. Samples were analysed as described in the Materials and methods section. Protein phosphorylation was monitored by autoradiography (bottom panel). (B) ΔUbl-Parkin activity is increased by wild-type TcPINK1. A 2 μg amount of full-length (WT) or ΔUbl-Parkin was incubated with 1 μg of wild-type (WT), kinase-inactive (KI) or no TcPINK1 in a kinase reaction for 60 min. The ubiquitylation reactions were then initiated by the addition of ubiquitylation assay components (E1, UbcH7 and FLAG–ubiquitin) and 2 μg of His6–SUMO-Miro1. Reactions were terminated after 60 min by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Miro1, ubiquitin (Ub) and Parkin were detected using anti-SUMO, anti-FLAG and anti-Parkin antibodies respectively. (C) Activation of ΔUbl-Parkin by TcPINK1 is abolished by S65A ubiquitin. ΔUbl-Parkin was incubated in the presence or absence of wild-type (WT) or kinase-inactive (KI) PINK1. The ubiquitylation reactions were then initiated by the addition of ubiquitylation assay components including 0.04 mM wild-type (WT) or S65A His6–FLAG–ubiquitin. Reactions were terminated after 60 min by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Miro1, ubiquitin and Parkin were detected using anti-SUMO, anti-FLAG and anti-Parkin antibodies respectively. The molecular mass in kDa is indicated.