Phosphorylation of Mitochondrial Polyubiquitin by PINK1 Promotes Parkin Mitochondrial Tethering (original) (raw)
Figure 1
Ub is a substrate of PINK1.
(A) Mass spectrometric screening of PINK1 substrates identified Ub phosphorylation at Ser65. Extracted ion chromatograms of dimethylated EpSTLHLVLR from mouse Ub phosphorylated by WT or KD PINK1 with or without CCCP treatment. Ub phosphorylation was specifically increased in PINK1-FLAG WT/_PINK1−/−_MEFs treated with CCCP. The peak area ratios of PINK1 WT+ CCCP to PINK1 WT – CCCP and PINK1 WT+ CCCP to PINK1 KD+ CCCP are 22.3 and 308.8, respectively. (B) Recombinant glutathione S-transferase (GST), GST-fusion Ub (Ub, WT and S65A) and GST-fusion Parkin Ubl domains were incubated with TcPINK1 WT or KD in 40 µl of kinase reaction buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 10 mM MgCl2, 2 mM DTT and 2 mM ATP) for 90 min at 30°C, and they were subsequently separated on a Phos-tag gel and a conventional polyacrylamide gel, followed by western blotting with anti-GST antibodies. (C) Alignment of the amino acid sequences surrounding Ser65 (marked by an arrow) from human Ub and human Parkin. The numbers correspond to the residue numbers in the Parkin and Ub proteins. Acidic, basic and hydrophobic amino acids are shown in red, green and grey, respectively. Amino acids with amido and hydroxyl groups are shown in blue and yellow, respectively. (D) Ser65 of Ub is the only site phosphorylated by PINK1. The GST, GST-Ub (WT, S65A, T66A and S65A/T66A) and Parkin Ubl domains were incubated with TcPINK1 WT or KD as in (A). The ATP source used was 10 µCi of γ-32P ATP. Phosphorylated Ub was detected by autoradiography (32P). The reaction mixtures were subjected to SDS-PAGE, CBB staining and autoradiography. (E) Both K48- and K63-linked polyUb chains were phosphorylated by PINK1. The kinase assay was performed as in (D). His-Ub; recombinant 6× His-tagged Ub.