Figure 1.

(Overleaf.) TcPINK1 phosphorylates human Parkin at Ser⁶⁵ in vitro. (a) The indicated PD-linked proteins (1 μM) were incubated with either full-length MBP-fusion of wild-type TcPINK1 (1–570) or kinase-inactive (KI) TcPINK1 (D359A) (0.5 μg) and [γ-³²P] ATP for 30 min. Assays were terminated by addition of SDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-³²P] ATP was detected by autoradiography (upper panel). Similar results were obtained in three independent experiments. Fine dividing lines indicate that reactions were resolved on separate gels. The substrate bands on the Coomassie gel are denoted with a small red asterisk. All substrates were of human sequence and expressed in E. coli unless otherwise indicated. Tags on the substrates used for this experiment were glutathione s-transferase (GST)-α-synuclein, Parkin (no tag as His-SUMO tag cleaved off), His-UCHL1, GST-DJ1, GST-LRRK2 KI (1326-end D2017A), MBP-ATP13A2, GST-Omi, MBP-PLA2G6, GST-FBX07, GST-GAK-kinase-inactive (D191A), VPS35 (no tag as GST-tag cleaved off). (b) As in (a) except that proteins reported to interact with PINK1 were tested as PINK1 substrates. Human DJ1, Omi, TRAP1, PARL, NCS1 and Miro2 were expressed in E. coli with an N-terminal GST tag. Similar results were obtained in three independent experiments. (c) Timecourse of phosphorylation of Parkin by wild-type TcPINK1. MBP-TcPINK1 (0.5 μg) was incubated in the presence of GST-Parkin (1 μg) and [γ-³²P] ATP for the times indicated and assays terminated by addition of SDS loading buffer. Samples were subjected to SDS-PAGE and proteins detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-³²P] ATP was detected by autoradiography (upper panel). Gel pieces were quantified by Cerenkov counting for calculation of the stoichiometry of Parkin phosphorylation. Similar results were obtained in two independent experiments. (d) Mapping of phosphopeptides on Parkin after phosphorylation by TcPINK1 in vitro. Full-length GST-Parkin (1 μg) was incubated with 2 μg of either wild-type TcPINK1 (1–570) or KI TcPINK1 (D359A) in the presence of Mg²⁺[γ-³²P] ATP for 60 min. Assays were terminated by addition of LDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining and phosphorylated Parkin was digested with trypsin. The resultant peptides were separated by reverse phase HPLC on a Vydac C₁₈ column (Vydac 218TP5215) equilibrated in 0.1% (v/v) trifluoroacetic acid and the column developed with an acetonitrile gradient (diagonal line). The flow rate was 0.2 ml min⁻¹ and fractions (0.1 ml each) were collected and analysed for ³²P radioactivity by Cerenkov counting. Two major ³²P-labelled peaks (P1, P2) were identified following incubation with wild-type TcPINK1 (left). No peaks were identified following incubation with kinase-inactive TcPINK1 (right). (e) Schematic of domain organization of Parkin illustrating that Ser⁶⁵ lies within the Ubl domain (upper panel) and sequence alignment of residues around Ser⁶⁵ in human Parkin and a variety of lower organisms showing high degree of conservation. Abbreviations: Ubl, ubiquitin-like; IBR, in-between-RING; RING, really interesting new gene. (f) Mutation of Ser⁶⁵Ala (S65A) abolishes Parkin phosphorylation by TcPINK1. Full-length wild-type TcPINK1 (1–570) and kinase inactive TcPINK1 (D359A) against wild-type or S65A mutants of full-length Parkin, or the isolated Ubl-domain-containing N-terminal fragment (residues 1–108). The indicated substrates (2 μM) were incubated in the presence of the indicated enzyme (1 μg) and [γ-³²P] ATP for 30 min. Assays were terminated by addition of SDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-³²P] ATP was detected by autoradiography (upper panel).