Ubiquitin is a direct substrate of TcPINK1 in vitro
(A) Sequence alignment of residues around Ser65 in human ubiquitin and a range of ubiquitin-like modifiers and Ubl-domain-containing human proteins. (B) Ubiquitin and Parkin are specific substrates of TcPINK1. The indicated ubiquitin-like modifiers and Ubl-domain-containing proteins (1 μg) were incubated with wild-type (WT) or kinase-inactive (KI) TcPINK1 and Mg2+-[γ-32P]ATP for 60 min. Assays were terminated by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Proteins were detected by Colloidal Coomassie Blue staining (top panel) and incorporation of [γ-32P]ATP was detected by autoradiography (bottom panel). All substrates were of human sequence and expressed in E. coli. Tags on the substrates used for this experiment were GST–OTU1, untagged Nedd8, untagged ISG15, His6–SUMO1-(1–97), ubiquilin2 (His6–SUMO tag cleaved off), His6–HOIL1 and USP4. Asterisks denote the correct substrate band. (C) TcPINK1 is a specific upstream kinase of ubiquitin. The indicated kinases (1 μg) were incubated with ubiquitin and Mg2+-[γ-32P]ATP for 60 min. Assays were terminated and analysed as described in (B). Except for PINK1, all kinases were of human sequence and expression tags used were GST–MLK1-(132–413), GST–CDK2-(2–298)/cyclin A2-(171–432); His6–IKKε-(1–716), His6–IKKβ-(1–716), His6–Aurora A-(2–403), His6–NUAK1-(2–660), His6–GSK3β-(2–420) and His6–PLK1-(1–603). Broken dividing lines indicate separate gels; asterisks denote the kinase band. The molecular mass in kDa is indicated.