Extended Data Fig. 9: Regulation of PINK1 activity by oxidation.
a, Extended sequence alignment indicating that Cys169 and Cys360 are well conserved in PINK1, and invariant in mammalian PINK1. Cys169 is a Thr in TcPINK1, and a Ser in many fish species. b, Comparison of PhPINK1 and TcPINK1, for their ability to be regulated by oxidation, shown in Phos-tag ubiquitin phosphorylation assays (see Fig. 5b). While PhPINK1 activity is abrogated with H2O2, TcPINK1 with Thr172 in the P-loop, remains active. The observed reduction in TcPINK1 activity could be a result of oxidation of the conserved Cys362 (Cys360 in PhPINK1) in the active site. Experiments were performed in biological triplicate. See Supplementary Fig. 1 for uncropped gel. c, Inhibition of PhPINK1 ubiquitin phosphorylation activity can be reversed with DTT, suggesting reversible regulatory oxidation. See Methods. Experiments were performed in biological triplicate. See Supplementary Fig. 1 for uncropped gel. d, Time course assessment of HsPINK1 Cys–Ala mutants transiently expressed in HeLa PINK1−/− cells. OA treatment leads to accumulation of HsPINK1 and slightly altered autophosphorylation for both C166A and C387A. The HsPINK1(C166A) mutant was almost completely deficient in ubiquitin phosphorylation, while HsPINK1(C387A) showed highly reduced but still detectable phosphorylated ubiquitin levels. Experiments were performed in biological triplicate. See Supplementary Fig. 1 for uncropped blots. e, Time course of HsPINK1 mutants as in d, but using stable HsPINK1 expression in the presence of YFP–Parkin. Presence of YFP–Parkin seemingly increases levels of phosphorylated ubiquitin for all HsPINK1 variants as compared to d, yet overall, phosphorylated ubiquitin and phosphorylated Parkin levels remain strongly diminished with HsPINK1(C166A), and to a lesser degree with HsPINK1(C387A). Experiments were performed in biological triplicate. See Supplementary Fig. 1 for uncropped blots. f, Translocation of YFP–Parkin (cyan) to mitochondria (magenta, TOM20–Halo) in HeLa PINK1−/− stably expressing HsPINK1 Cys–Ala variants upon OA treatment, imaged using lattice light sheet microscopy. Maximum intensity projections are shown for four different timepoints. YFP–Parkin translocation is delayed in cells expressing either HsPINK1(C166A) or HsPINK1(C387A) relative to WT hHsPINK1. A kinase dead (KD) HsPINK1 variant was included as a control. Images are representative of three independent experiments. Scale bar is 10 μm. See Supplementary Video 1. g, Quantification of YFP–Parkin translocation in f. The cumulative fraction of cells exhibiting YFP–Parkin translocation is shown over time. Approximately 70 cells were counted per cell line. Each curve was fitted to determine the time for 50% of the cells to feature translocation. Significant differences between curves were determined using a two-sample Kolmogorov–Smirnov test. A MATLAB script and Source Data are available as Supplementary Material. Exact p values are: WT–C166A: p < 2.83 × 10−20; WT–C387A: p < 2.36 × 10−18. h, HsPINK1 mutants expressed in HeLa PINK1−/− cells and treated with OA for 2 h. Activity of HsPINK1(C166A) and HsPINK1(C166S) can be partially restored by an additional S167N mutation, mimicking the sequence observed in many fish species. A TcPINK1-like sequence introduced into HsPINK1, C166T/S167N, is less active than the HsPINK1(C166S/S167N) mutant. The additional Asn in the P-loop of the HsPINK1(C166S/S167N) mutant recovers activity of HsPINK1(C166S), suggesting that both residues are also important in ubiquitin/Ubl substrate interactions, and not merely involved in dimerization. Experiments were performed in biological triplicate. See Supplementary Fig. 1 for uncropped blots. i, A redox active switch in fructosamine-3-kinases (PDB: 6OID) is conceptually similar, utilizing a Cys at an identical position (Cys32) for Cys-mediated cross-linking and regulation of kinase activity by oxidation36, however the overall orientation of kinase domains is dissimilar.
